Transplants for myocardial scars

ABSTRACT

A method is provided for forming a graft in heart tissue which comprises the transplantation of cells chosen from cardiomyocytes, fibroblasts, smooth muscle cells, endothelial cells and skeletal myoblasts. The grafts are especially useful in treating scar tissue on the heart.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/634,304, filedAug. 8, 2000, which is a continuation of U.S. Ser. No. 09/099,994, filedJun. 19, 1998 (now U.S. Pat. No. 6,099,832)

FIELD OF THE INVENTION

The present invention relates to novel methods of cell transplantationinto scar tissue in the heart in order to improve heart function,stimulate angiogenesis, and to salvage myocardium. The invention alsorelates to the preparation and culturing of the subject cells prior totransplantation, a mechanism for the delivery of gene therapy using suchtransplants, and to grafts comprising such cells.

BACKGROUND OF THE INVENTION

Organ transplantation and surgical resection have been used to replaceor remove diseased non-functional myocardial tissue. Recently, fetalcellular transplantation has been used to improve neurologicaldeficiencies found in Parkinson's disease (Tompson, L. et al., Science257:868-870, 1992). In a similar approach, normal myoblasts have beentransplanted into the skeletal muscle of patients with Duchenne musculardystrophy (Gussoni, E. et al., Nature 356:435-438, 1992), where thetransplanted cells expressed dystrophin.

Fetal ventricular cardiomyocytes, atrial tumor cells, and skeletalmyoblasts have been transplanted into normal myocardium (Koh, G Y etal., Journal of Clinical Investigation 92:1548-54, 1993; Soonpaa, M H etal., Science 264:98-101, 1994; U.S. Pat. No. 5,602,301). In the studiesdescribed in these references, the cells were transplanted into themiddle and thickest layer of the heart, composed of cardiac muscle,which has an excellent blood supply. Transplanted atrial tumor cellsformed intercalated disc junctions with the host cardiomyocytes.Myocardial function was not assessed.

Cardiac scar tissue is formed after the ventricular wall of the heartnecroses due to damage. In contrast to myocardial tissue, cardiac scartissue contains no cardiac muscle cells. Instead, it is composed ofconnective tissue cells, such as fibroblasts, and non-cellularcomponents, such as collagen and fibronectin. Cardiac scar tissue isnon-contractile, and, therefore, interferes with normal cardiacfunction. Mature scar tissue is thought to be an inert tissue having alimited blood supply. Accordingly, the prior art suggests that culturedcells could not be successfully transplanted into mature scar tissue.

Scar tissue is much thinner than normal myocardium. In the method taughtby Field in U.S. Pat. No. 5,602,301, cellular grafts are introduced intothe myocardium by injection. However, this method, if applied to themuch thinner scar tissue, would result in tissue ballooning and anaccompanying increase in pressure within the region of cell injection.As a result, the transplanted cellular material would leak from thepuncture point of the injection needle upon withdrawal, and theefficiency of such transplants would be reduced.

Thus, there is a need to develop cellular allo- and autotransplantationtechnology within scar tissue of the diseased myocardium to improvecontractile function, minimize myocardial remodeling, stimulateangiogenesis, deliver gene therapy, rebuild the heart, and salvagedamaged cardiomyocytes. The present invention addresses these needs.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide cell transplantationmethods for treating scar tissue in the myocardium which overcomedeficiencies in the prior art The invention illustrates that atrialmyocytes, smooth muscle cells, endothelial cells, and fibroblasts can besuccessfully transplanted into the scar tissue formed after ventricularnecrosis and into tissue membranes and porous synthetic membranes. Thecell grafts form tissue that survived the three month duration of thestudy, improved myocardial function, limited myocardial remodeling, andstimulated angiogenesis. The presence of the grafts did not induce overtcardiac arrhythmias. When auto-cell transplantation occurred,immunorejection did not occur.

In a first aspect, the invention features a method of forming a stablemyocardial graft in a mammal comprising, transplanting cells intomyocardial tissue or scar tissue in the heart. Cells are chosen from thegroup consisting of: adult cardiomyocytes, fetal cardiomyocytes,pediatric cardiomyocytes, adult fibroblasts, fetal fibroblasts, smoothmuscle cells, endothelial cells, and skeletal myoblasts.

In preferred embodiments of the first aspect of the invention, cells maybe chosen from adult or fetal smooth muscle cells and fibroblasts, adultcardiomyocytes and endothelial cells may be co-transplanted, adultcardiomyocytes may be derived from atrial tissue, the graft may bederived from auto-, allo- or xenotransplantation, and the graft maycomprise adult cardiomyocytes derived from autotransplantation, such ascardiomyocytes derived from atrial tissue.

In another preferred embodiment of the first aspect of the invention,the cells may be directly introduced into the myocardial tissue or thescar tissue, for example, by injection, and the injection site may besealed with a biological adhesive to prevent leakage of the cells.

In other preferred embodiments of the first aspect of the invention thecells may be suspended on a biodegradable or non-degradable mesh, or maybe transfected to deliver recombinant molecules to the myocardial tissueor the scar tissue.

In still another embodiment of the first aspect of the invention, thecells may be used in myocardial reconstructive surgery, and may beattached to the outer surface of the myocardial tissue or the scartissue with a biological adhesive, or may be transplanted following aninflammatory response in the myocardial tissue. In addition, growthfactors may be co-transplanted with the cells. Growth factors are chosenfrom the group consisting of: insulin-like growth factors I and II;transforming growth factor-β1, platelet-derived growth factor-B, basicfibroblast growth factor, and, vascular endothelial growth factor.

In yet other preferred embodiments of the first aspect of the invention,the cells are transplanted into scar tissue, and at least 10%, 20%, or30% of the scar tissue area is occupied by transplanted cells four weeksafter transplantation.

In a second aspect, the invention features a therapeutic graft forapplication in mammalian myocardial tissue or scar tissue in the heart,comprising transplanted cells chosen from the group consisting of: adultcardiomyocytes, pediatric cardiomyocytes, fetal cardiomyocytes, adultfibroblasts, fetal fibroblasts, adult smooth muscle cells, fetal smoothmuscle cells, endothelial cells, and skeletal myoblasts.

In preferred embodiments of the second aspect of the invention, thegraft may comprise adult cardiomyocytes and endothelial cells, thetransplanted cells may be chosen from smooth muscle cells and fetalfibroblasts, the adult cardiomyocytes may be derived from atrial tissue,or the graft may be derived from auto-, allo- or xenotransplantation.The graft may comprise adult cardiomyocytes derived fromautotransplantation and the cardiomyocytes may be derived from atrialtissue. The cells of the graft may be introduced into myocardial tissueor scar tissue by injection, and the cells may be transfected to deliverrecombinant molecules to myocardial tissue or scar tissue. The graft mayfurther comprise growth factors, for example, insulin-like growthfactors I and II, transforming growth factor-β1, platelet-derived growthfactor-B, basic fibroblast growth factor, and, vascular endothelialgrowth factor. Cells of the graft also may be suspended on a mesh (e.g.,a biodegradable mesh).

In a third aspect, the invention features a therapeutic graft, forimplantation into mammalian myocardial tissue or scar tissue in theheart, comprising a suitable biodegradable or non-biodegradablescaffolding having cells supported thereon. The cells are chosen fromthe group consisting of: adult cardiomyocytes, pediatric cardiomyocytes,fetal cardiomyocytes, adult fibroblasts, fetal fibroblasts, smoothmuscle cells (e.g, adult smooth muscle cells or fetal smooth musclecells), endothelial cells, and skeletal myoblasts.

In preferred embodiments of the third aspect of the invention, adultcardiomyocytes may be derived from atrial tissue, and the graft maycomprise adult cardiomyocytes and adult endothelial cells. The graft maybe used in cardiomyoplasty. The scaffolding of the graft may compriseDacron or polyglycolic acid polymers with or without polylactic acidpolymers, the cellular material may consist of cardiomyocytes, smoothmuscle cells or endothelial cells, and the graft may further include animplantable pacemaker.

Grafts according to the third aspect of the invention may be used forclosing cardiac defects, and for myocardial reconstructive surgery.

In a fourth aspect, the invention features a method of culturingcardiomyocytes from pediatric mammalian myocardial tissue comprising: a)comminuting said myocardial tissue; b) digesting said tissue for 15minutes in a digesting solution containing 0.2% trypsin and 0.1%collagenase dissolved in phosphate buffered saline and separating thedigested tissue solution from the remaining myocardial tissue; c) addingto the digested tissue solution a culture medium comprising Iscove'smodified Dulbecco's medium (IMDM), 10% fetal bovine serum, and 0.1 mMβ-mercaptoethanol; culture medium being added in a ratio of 20 volumesof culture medium to 1 volume of digesting solution; d) centrifuging theresulting solution at 581×g for 5 minutes and discarding thesupernatant; e) re-suspending the pellet in fresh culture medium; f)culturing the suspension in 10% fetal bovine serum and 0.1 mMβ-mercaptoethanol; and, g) isolating cardiomyocytes from the culture.

In preferred embodiments of the fourth aspect of the invention themethod may further include passaging cardiomyocytes by sub-culturingwith a sub-culturing enzyme solution comprising 0.01% trypsin, 0.02%glucose, and 0.5 mM EDTA. The method of the fourth aspect may furtherinclude storing the cardiomyocytes by a) dissociating culturedcardiomyocytes from the culture plate using sub-culturing enzymesolution; b) adding culture medium in a ratio of 5 volumes of culturemedium to 1 volume of sub-culturing enzyme solution; c) centrifuging thesolution at 581×g for 5 minutes; d) discarding the supernatant andre-suspending the pellet in 1 mL IMDM containing 20% fetal bovine serumand 20% glycerol; and, e) freezing and storing the resulting suspensionin liquid nitrogen. The method may further include thawing the frozensample at 37° C. and culturing the cardiomyocytes for 3 to 5 days in asolution of IMDM containing 20% fetal bovine serum.

In a fifth aspect, the invention features a method of culturingcardiomyocytes from adult mammalian myocardial tissue comprising: a)comminuting said myocardial tissue; b) digesting the tissue for 15minutes in a digesting solution containing 0.2% trypsin and 0.1%collagenase dissolved in phosphate buffered saline; c) separating thedigested tissue solution and digesting the remaining tissue with freshdigesting solution for 10 minutes; d) combining both digested tissuesolutions from steps (b) and (c) and adding a culture medium comprisingIscove's modified Dulbecco's medium (IMDM, containing 10% fetal bovineserum, and, 0.1 mM β-mercaptoethanol) in a ratio of 20 volumes ofculture medium to 1 volume of said digesting solution; e) centrifugingthe resulting solution at 581×g for 5 minutes and discarding thesupernatant; f) re-suspending the pellet in fresh culture medium; g)culturing the suspension in 10% fetal bovine serum and 0.1 mMβ-mercaptoethanol; and, h) isolating cardiomyocytes from the culture.

In a preferred embodiment of the fifth aspect of the invention, themethod may further include passaging the cardiomyocytes usingsub-culturing enzyme solution comprising 0.01% trypsin, 0.02% glucose,and 0.5 mM EDTA.

The method of the fifth aspect also may further include storingcardiomyocytes by a) dissociating cultured cardiomyocytes from theculture plate using sub-culturing enzyme solution; b) adding culturemedium in a ratio of 5 volumes of culture medium to 1 volume ofsubculturing enzyme solution; c) centrifuging the solution at 581×g for5 minutes; d) discarding the supernatant and re-suspending the pellet in1 mL IMDM containing 20% fetal bovine serum and 20% glycerol; and, e)freezing and storing the resulting suspension in liquid nitrogen.

In another embodiment of the fifth aspect, the method may furtherinclude thawing the frozen sample at 37° C. and culturing thecardiomyocytes for 3 to 5 days in a solution of IMDM containing 20%fetal bovine serum.

In a sixth aspect, the invention features a method of treatingdefective, damaged or scarified heart tissue, comprising transplantinginto the tissue a graft of cells chosen from the group consisting of:adult cardiomyocytes, pediatric cardiomyocytes, fetal cardiomyocytes,adult fibroblasts, fetal fibroblasts, adult smooth muscle cells, fetalsmooth muscle cells, endothelial cells, and skeletal myoblasts.

In preferred embodiments of the sixth aspect of the invention, the adultcardiomyocytes may be derived from atrial tissue, cells in the graft maybe adult cardiomyocytes and endothelial cells, the cells may be directlyintroduced into heart tissue, and the graft may be a patch comprisingcells suspended on a biologically acceptable biodegradable ornon-biodegradable scaffolding.

In still other preferred embodiments of the sixth aspect of theinvention, the cells are transplanted into scar tissue, and at least10%, 20%, or 30% of the scar tissue is occupied by transplanted cellsfour weeks after transplantation.

In another preferred embodiment of the sixth aspect of the invention,the method may comprise the steps of: (a) surgically removing defectiveheart tissue thereby creating an opening; and, (b) attaching the graftto the opening to form a water tight seal.

In a seventh aspect, the invention features isolated cells fortransplantation into myocardial scar tissue, selected from the groupconsisting of: adult cardiomyocytes, pediatric cardiomyocytes, adultfibroblasts, fetal fibroblasts, adult smooth muscle cells, fetal smoothmuscle cells, endothelial cells, and skeletal myoblasts, wherein thecells survive in myocardial scar tissue after transplantation andimprove cardiac function, relative to cardiac function of a heart havingsimilar myocardial scar tissue that is not transplanted with cells.Cardiac function is assessed by at least one of the criteria in thegroup consisting of: area occupied by scar tissue; vascularization ofscar tissue; blood flow to scar tissue; developed pressure, systolicpressure; end diastolic pressure; and dp/dt.

In preferred embodiments of the seventh aspect of the invention, thecells may comprise at least two of the cell types selected from thegroup. For example, the cells may comprise a combination of: adultcardiomyocytes and endothelial cells; pediatric cardiomyocytes andendothelial cells; or myoblasts and endothelial cells.

In an eighth aspect, the invention features a method for testing apharmacological agent that is intended to prevent or ameliorate cardiacdamage during cardiac surgery. The method comprises exposing thepharmacological agent to isolated cells selected from the groupconsisting of: adult cardiomyocytes, pediatric cardiomyocytes, adultfibroblasts, fetal fibroblasts, adult smooth muscle cells, fetal smoothmuscle cells, endothelial cells, and skeletal myoblasts, wherein thecells survive in myocardial scar tissue after transplantation andimprove cardiac function, relative to cardiac function of a heart havingsimilar myocardial scar tissue that is not transplanted with cells(cardiac function is assessed by at least one of the criteria in thegroup consisting of: area occupied by scar tissue; vascularization ofscar tissue; blood flow to scar tissue; developed pressure, systolicpressure; end diastolic pressure; and dp/dt), wherein cells exposed tothe pharmacological agent prevent or ameliorates cardiac damage duringcardiac surgery, compared to cells not exposed to the pharmacologicalagent.

In an ninth aspect, the invention features a method of forming a stablecardiac graft in a mammal, comprising transplanting into the scar tissueof a heart, cells chosen from the group consisting of: adultcardiomyocytes; pediatric cardiomyocytes; adult fibroblasts; fetalfibroblasts; adult smooth muscle cells; fetal smooth muscle cells;endothelial cells; and skeletal myoblasts, wherein the cells survive inscar tissue in a heart after transplantation into scar tissue, andwherein the cells improve cardiac function, relative to cardiac functionof a heart having similar myocardial scar tissue that is nottransplanted with such cells, wherein cardiac function is assessed by atleast one of the criteria in the group consisting of: area occupied byscar tissue; vascularization of scar tissue; blood flow to scar tissue;developed pressure, systolic pressure; end diastolic pressure; anddp/dt, wherein at least 10% of scar tissue is occupied by transplantedcells four weeks after transplantation.

In other embodiments of the ninth aspect of the invention, at least 20%or at least 30% of the scar tissue may be occupied by transplanted cellsfour weeks after transplantation, or at least 40% or at least 50% of thescar tissue may be occupied by transplanted cells eight weeks aftertransplantation.

In preferred embodiments of the ninth aspect, the cells may include atleast two types of cells selected from the group. For example, the cellsmay comprise a combination of: adult cardiomyocytes and endothelialcells; pediatric cardiomyocytes and endothelial cells; or myoblasts andendothelial cells.

In other preferred embodiments of the ninth aspect of the invention,growth factors are co-transplanted with the cells. The growth factorsare chosen from the group consisting of: insulin-like growth factors Iand II; transforming growth factor-β1; platelet-derived growth factor-B;basic fibroblast growth factor; and, vascular endothelial growth factor.

In a tenth aspect, the invention features a method of treatingdefective, damaged or scarified heart tissue, comprising transplantinginto defective, damaged or scarified heart tissue a graft of cells,wherein the graft of cells comprises a combination of: adultcardiomyocytes and endothelial cells; pediatric cardiomyocytes andendothelial cells; or myoblasts and endothelial cells.

In preferred embodiments of the tenth aspect of the invention, the graftmay be used for cardiomyoplasty, for closing cardiac defects, or formyocardial reconstructive surgery.

In an eleventh aspect, the invention features a therapeutic graft forimplantation in mammalian myocardial tissue or scar tissue in a heart,comprising biodegradable or non-biodegradable scaffolding supportingcells, wherein the cells consist of a combination of: adultcardiomyocytes plus endothelial cells; pediatric cardiomyocytes plusendothelial cells; or myoblasts plus endothelial cells.

In various embodiments of the eleventh aspect of the invention, thescaffolding comprises Dacron or polyglycolic acid polymers with orwithout polylactic acid polymers, or further includes an implantablepacemaker.

In another embodiment of the eleventh aspect, the cells may betransfected to deliver recombinant molecules to the myocardial tissue orscar tissue.

In still another embodiment of the eleventh aspect, the graft maycomprise growth factors, for example: insulin-like growth factors I andII; transforming growth factor-β1; platelet-derived growth factor-B;basic fibroblast growth factor: and, vascular endothelial growth factor.

In yet another embodiment of the eleventh aspect, the cells may besuspended on a biodegradable mesh.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the present invention will become moreapparent in the following detailed description wherein references aremade to the following figures:

FIG. 1 is a graph showing the improvement in myocardial function ofscarred hearts having smooth muscle cells transplanted into the scars.

FIG. 2 is a graph showing the amount of blood flow (represented asmicrosphere count) to control scar tissue and scar tissue transplantedwith endothelial cells.

FIG. 3 is a graph showing systolic pressure of transplantation andcontrol hearts with increasing balloon volume.

FIG. 4 is a graph showing developed pressure of transplantation andcontrol hearts with increasing balloon volume.

FIG. 5 is a diagram showing the design of cardiaccryo-injury/endothelial cell transplantation experiments. Rats weresacrificed at 1, 2, 4 and 8 weeks (wks) after cryo-injury of the leftventricular free wall (LVFW) for the histological study of theremodeling of the left ventricle. After cryo-injury of the LVFW theanimals were randomly divided into three groups (group 1, 2 and 3). Ineach group, half of animals were transplanted with cardiomyocytes(transplant) and half animals were transplanted with cultured medium(control). Transplantation into the center of the LVFW scar wasperformed immediately (group 1), 2 weeks (group 2) and 4 weeks (group 3)after myocardial injury. All the animals were sacrificed at 8 weeksafter cryo-injury for histological and functional studies.

FIG. 6(A-D) shows photomicrographs of haematoxylin and eosin (H &E)-stained LVFW after cryo-injury. A) immediately after cryo-injury(magnification=200×), and B) 1 week (magnification=100×), C) 2 weeks(magnification 100×) D) 4 weeks (magnification=200×) after cryo-injury.The myocardium is fragmented immediately after cryo-injury. At 1 week, apredominantly mononuclear infiltrate is present and most of the necrosedcardiomyocytes have disappeared. At 2 weeks, fibroblasts and collagenare evident. The inflammatory infiltrate is almost gone. At 4 weeks, atransmural scar has formed.

FIG. 7(A-D) shows photomicrographs of H & E-stained heart sections (5×)at A) 1, B) 2, C) 4, and D) 8 weeks after cryo-injury at LVFW. The sizeof the scar tissue (indicated by arrows) increases with time afterinjury.

FIG. 8 is a graph showing percentages of scar tissue in the LVFW at 1week, 2 weeks, 4 weeks, and 8 weeks after cryo-injury. The scar tissuesize increased with time. Open circles represent individual scar sizes,and closed squares represent mean scar sizes±SE (N=7). ** p<0.01.

FIG. 9(A-D) shows photomicrographs of H & E-stained heart sections (5×)at 8 weeks after cryo-injury of the LVFW. The control heart (A) wastransplanted with culture medium. Experimental hearts were transplantedwith cultured fetal rat cardiomyocytes immediately (B), at 2 weeks (C)and at 4 weeks (D) after myocardial injury. Transplanted cardiac tissuewas not observed in (A) and (B) hearts. Transplanted tissue (indicatedby arrows) was found in myocardial scar tissue in (C) and (D) hearts.Host myocardium was usually present beneath the scar when the transplantwas present, but never when the transplant was absent. We attribute thepresence of the host myocardium to the cell transplant limiting scarexpansion.

FIG. 10 is a graph showing percentages of scar tissue and transplantedcardiac tissue in the LVFW at 8 weeks after cryo-injury. Fetal ratcardiomyocytes transplanted at 2 and 4 weeks after myocardial injuryformed cardiac tissues which occupied 34% and 31% of the total scararea, respectively. The cardiac transplant limited scar expansion.Results are expressed as mean±SE. ** p<0.01.

FIG. 11 shows a photomicrograph of fetal rat cardiomyocytes 6 weeksafter transplantation (100×). The transplanted tissue stained positively(dark-brown color) for myosin heavy chain using theavidin-biotin-peroxidase complex technique.

FIG. 12 shows graphs of systolic, diastolic, developed balloon pressuresand positive dp/dt of control and transplanted hearts with increasingballoon volumes. Fetal rat cardiomyocytes were transplanted immediatelyafter myocardial injury. There were no differences in systolic anddeveloped pressures between the transplanted and control hearts. Resultsare expressed as mean±SE (N=8). Closed circles represent control hearts;open squares represent transplanted hearts.

FIG. 13 shows graphs of systolic, diastolic, developed balloon pressuresand positive dp/dt of control and transplanted hearts with increasingballoon volumes. Transplantation was performed at 2 weeks aftermyocardial injury. Systolic and developed pressures of the transplantedrat hearts were greater (p<0.01) than those of the control hearts.Results are expressed as mean±SE (N=8). Closed circles represent controlhearts; open squares represent transplanted hearts.

FIG. 14 shows graphs of systolic, diastolic, developed balloon pressuresand positive dp/dt of control and transplanted hearts with increasingballoon volumes. Transplantation was performed at 4 weeks aftermyocardial injury. Systolic and developed pressures of the transplantedrat hearts were significantly greater than those of control hearts(p<0.01). Results are expressed as mean±SE (N=12). Closed circlesrepresent control hearts; open squares represent transplanted hearts.

FIG. 15 is a graph showing developed pressures at balloon volumes of0.04, 0.1, 0.2 and 0.3 ml of the hearts transplanted with fetalcardiomyocytes at 2 and 4 weeks after myocardial injury. The developedpressures of hearts transplanted at 2 weeks were significantly higherthan those of the hearts transplanted at 4 weeks. Results are expressedas mean±SE. * p<0.05, ** p<0.01.

FIG. 16 is a graph showing developed pressure in cryo-injured heartscontaining transmural scars transplanted with medium (control), adultcardiomyocytes (CM) or adult cardiomyocytes plus vascular endothelialcells (CM+EC).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

We have discovered methods for improving the outcome of patients havingmyocardial scars. Our discovery was made while investigating the use ofgrafts, comprising cellular transplants, for treating scar tissue in theheart. We transplanted cultured cells into the center of a matureventricular scar so that there was no contact between the transplantedcells and the host cells. Such methodology was followed to aid in theidentification of the transplant. Initially, in transplanting fetalcardiomyocytes into the mature scar, it was surprising to findstimulation of angiogenesis in the affected area and an improvement inheart function. Since we were previously unsuccessful in transplantingadult ventricular cardiomyocytes into myocardial scar tissue, we weresurprised that adult atrial cardiomyocytes could be successfullytransplanted into such scar tissue. In further studies, we havetransplanted auto- and allo-smooth muscle cells, fibroblasts, andendothelial cells into the center of a mature ventricular scar. As withour previous findings, the cell transplants formed stable grafts in thescar, improved myocardial function, decreased myocardial remodeling,stimulated angiogenesis, salvaged myocardium, and provided a means ofgene delivery. On the basis of these studies we expect that similarresults can be obtained from the use of skeletal myoblasts as well.

We have also developed a novel method for culturing, passaging andstoring postnatal (adult or pediatric) human ventricular cardiomyocytes.Transplantation of cultured postnatal cardiomyocytes provides anapproach for restoring function and blood flow to regions of the heartthat are destroyed by acquired heart disease or are absent because ofcongenital heart disease. The postnatal cardiomyocyte cell culturemethod further provides a model for testing a variety of pharmacologicalor other approaches for preventing or ameliorating cardiac damage duringcardiac surgery. Therefore, this unique method offers the promise ofpreventing injury as well as restoring heart function after cardiacinjury.

The following examples will assist those skilled in the art to betterunderstand the invention and its principles and advantages. It isintended that these examples be illustrative of the invention and notlimit the scope thereof.

General Methods

A) Cell Isolation, Culture, and Identification

The following procedures were approved by the hospital's ExperimentationCommittee.

1. Fetal Rat Cardiomyocyte Cultures: Fetal rat cardiomyocytes wereisolated using an enzymatic digestion method (Li, R-K et al.,Circulation Research 78:283-288, 1996; Li, R-K et al., Annals ofThoracic Surgery 62:654-661, 1996) from 18-day gestationalSprague-Dawley rat heart ventricles (Charles River Canada Inc. Quebec,Canada). Fetal rats were anesthetized with intraperitoneal injection ofsodium pentobarbital (30 mg/kg) and the hearts were then excised. Theheart tissues were washed with phosphate-buffered saline (NaCl 136.9 mM,KCl 2.7 mM, Na₂HPO₄ 8.1 mM, KH₂PO₄ 1.5 mM, pH 7.3). The tissues wereminced and incubated in 10 ml phosphate buffered saline containing 0.2%trypsin, 0.1% collagenase, and 0.02% glucose for 30 minutes at 37° C.The cardiomyocytes were then isolated by repetitive pipetting of thedigested myocardial tissue. The cells in the supernatant weretransferred to a tube containing 20 ml of cell culture medium (Iscove'smodified Dulbecco's medium containing 10% fetal bovine serum, 0.1mmole/L β-mercaptoethanol, 100 units/ml penicillin and 100 ug/mlstreptomycin). The tube was centrifuged at 600×g for 5 minutes at roomtemperature and the cell pellet was resuspended in the cell culturemedium for purification by plating (see Section 10 below).

2. Adult Rat Ventricular Cardiomyocyte Cultures: Adult rats (CharlesRiver Canada Inc. Quebec, Canada) were anesthetized with intramuscularadministration of ketamine hydrochloride (22 mg/kg body weight) followedby an intraperitoneal injection of sodium pentobarbital (30 mg/kg) andthe hearts were then excised. Cardiomyocytes were isolated by enzymaticdigestion as described in Section 1. Adult human ventricular biopsiesobtained from the operating theatre were similarly enzymaticallydigested.

3. Adult Rat Atrial Cardiomyocyte Cultures: Adult rats weighing 400 g(Charles River Canada Inc. Quebec, Canada) were anesthetized as inSection 2 above. The atrial appendages were ligated and removed (ratssurvive this procedure), after which the atrial tissue was used forcardiomyocyte isolation as described in Section 1. Adult human atrialtissue was obtained from the operating theatre and similarlyenzymatically digested.

4. Fetal Rat Smooth Muscle Cells: Fetal rat smooth muscle cells wereisolated from 18-day gestational Sprague-Dawley rat stomachs byenzymatic digestion (Li, R-K et al., Journal of Tissue Culture andMethods 14:93-100, 1992). Fetal rats (Charles River Canada Inc. Quebec,Canada) were anesthetized with pentobarbital (30 mg/kg body weight,intraperitoneal) and the stomachs were then excised. The stomachs werewashed with phosphate buffered saline, minced, and incubated in 10 mlphosphate buffered saline containing 0.2% trypsin, 0.1% collagenase, and0.02% glucose for 30 minutes at 37° C. Smooth muscle cells were isolatedby repetitive pipetting of the digested stomach tissue. The cells in thesupernatant were transferred to a tube containing 20 ml of cell culturemedium (199 medium containing 20% fetal bovine serum, 100 units/mlpenicillin and 100 ug/ml streptomycin), and centrifuged at 600×g for 5minutes at room temperature, after which the cells were resuspended inthe cell culture medium and cultured.

5. Adult Rat Smooth Muscle Cells: Adult female rats (Charles RiverCanada Inc. Quebec, Canada) were anesthetized as described in Section 2above. Uteri were removed after occlusion of blood vessels and incisionwas closed. Smooth muscle cells were isolated from uterus as describedin Section 4.

6. Fetal Rat Fibroblasts: Fetal rat skin biopsies were obtained fromanesthetized 18-day gestational Sprague-Dawley rats, and fibroblastsfrom fetal rat skin were isolated, purified and cultured as previouslydescribed (Mickle et al., Journal of Molecular and Cell Cardiology22:1297-1304, 1990). Briefly, the tissue was washed with phosphatebuffered saline, minced, and digested for 30 minutes at 37° C. in 10 mlphosphate buffered saline containing 0.2% trypsin, 0.1% collagenase, and0.02% glucose. Fibroblasts were isolated by repetitive pipetting of thedigested skin tissue. The cells in the supernatant were transferred intoa tube containing 20 ml of cell culture medium (Dulbecco's ModifiedEssential Medium containing 10% fetal bovine serum, 100 units/mlpenicillin and 100 ug/ml streptomycin) and centrifuged at 600×g for 5minutes at room temperature, after which the cells were resuspended inthe cell culture medium and cultured.

7. Adult Rat Fibroblasts: Skin biopsies were obtained from anesthetizedadult Sprague-Dawley rats. Fibroblasts were isolated and cultured asdescribed in Section 6.

8. Adult Rat Endothelial Cells: Adult rat vascular endothelial cellswere isolated from Sprague-Dawley rat aorta by enzymatic digestion(Mickle et al., Journal of Molecular and Cell Cardiology 22:1297-1304,1990). Adult rats (Charles River Canada Inc. Quebec, Canada) wereanesthetized as described in Section 2. Aortas were excised, washed withphosphate buffered saline, incubated for 30 minutes at 37° C. in 10 mlphosphate buffered saline containing 0.2% trypsin, 0.1% collagenase, and0.02% glucose, and washed with cell culture medium (199 mediumcontaining 20% fetal bovine serum, 100 units/ml penicillin and 100 ug/mlstreptomycin), after which the isolated endothelial cells were cultured.

9. Human Cell Isolation, Culturing and Storage: Human cardiomyocyteswere isolated from atrial appendages and ventricular myocardial biopsiesobtained from patients undergoing corrective cardiac surgery. Humanmyocardium was dissected to remove connective tissue and then minced topieces less than 1 mm³ in size.

9(a). Pediatric Cardiomyocytes: Pediatric tissue was digested for 15minutes in an enzymatic digestion solution containing 0.2% trypsin, 0.1%collagenase dissolved in phosphate-buffered saline (no calcium or EDTAwas added). Culture medium containing Iscove's modified Dulbecco'smedium (IMDM), 10% fetal bovine serum and 0.1 mM β-mercaptoethanol wasadded in a ratio of 20 volumes culture medium to 1 volume enyzmaticdigestion solution. The cell suspension was centrifuged at 581 g for 5minutes, after which the supernatant was discarded. The cell and tissuepellet was resuspended in culture medium, after which the isolated cellswere cultured on a dish for 5 to 8 days. Cardiomyocytes that migratedfrom this culture were collected by a Pasteur pipette and cultured onceagain.

9(b). Adult Cardiomyocytes: In contrast to pediatric myocardiumdigestion, adult human myocardium was digested twice. The seconddigestion was necessary for the adult tissue due to the increased amountof connective tissue. After the first digestion (as described in Section9a), the cell suspension was collected, after which the tissue wasre-digested for 10 minutes and the second cell suspension was collected.The two collected suspensions were combined, and centrifuged, afterwhich the cells were resuspended and cultured. The digested tissuefragments in the pellet were collected and cultured for no longer than 2days (to avoid cell deterioration) and a further enzymatic digestioncarried out on the remaining undigested tissue if sufficient cells werenot found in the suspensions. In addition, cardiomyocytes that migratedfrom the cultured tissue fragments were isolated and re-cultured.

9(c). Passaging of Cultures: The cardiomyocytes were cultured in amedium containing IMDM, 10% fetal bovine serum and 0.1 mMβ-mercaptoethanol. The subculturing enzymatic solution for celldissociation contained 0.01% trypsin, 0.02% glucose and 0.5 mM EDTA. Thecells were subcultured when the culture reached confluence (i.e. whenthe cell culture covered the dish surface and the cells began contactingeach other). Cells that were subcultured before reaching confluencyrapidly became de-differentiated. Such de-differentiated cells cannot besuccessfully transplanted. If the cells were allowed to becomeover-confluent, enzymatic digestion yielded cell clumps that would notdissociate. Cardiomyocytes in cell clumps did not divide in culture.

9(d). Storage of Cultured Cells: Primary cultures of humancardiomyocytes were dissociated from culture plates using thesubculturing enzymatic digestion solution, after which culture mediumwas added in a ratio of 5 volumes culture medium to 1 volume digestionsolution, and the cell suspension was centrifuged at 581 g for 5minutes. The supernatant was removed and the cell pellet was gentlyre-suspended in 1 mL IMDM containing 20% fetal bovine serum and 20%glycerol, transferred to a sterile cryo-vial, and placed in a Nalgenefreezing container containing isopropranol in its base. Cryo-vials werestored in a −80° C. freezer container for a period of time that: (a)ensured that the cells reached −80° C.; and (b) prevented over-oxidationof the cells. In the present example, cryo-vials were stored for aminimum of 4 hours and no longer than 8 hours. Cryo-vials containing thecells were placed in liquid nitrogen for long term storage.

9(e). Thawing of Cultures: When the stored cells were to be cultured,the vial was removed from liquid nitrogen and warmed at 37° C. Although37° C. was preferred, cells were also thawed at other temperatures thatallowed for rapid yet non-destructive warming. The initial plating ofthe cells was done in 10 ml of IMDM medium containing 20% fetal bovineserum (warming medium). Cells were kept in warming medium for 3 to 5days to allow the cells to attach firmly to the culture dish beforeswitching to the usual culture medium. The warming medium must containIMDM and 20% fetal bovine serum. For example, if the warming mediumcontains 10% rather than 20% fetal bovine serum, the humancardiomyocytes will not divide and will de-differentiate.De-differentiated cardiomyocytes cannot be used for transplantation.

Cardiomyocytes that were cryo-frozen and warmed as described weresuccessfully subcultured and were morphologically identical to cellsthat had not been frozen. Cells that had been frozen and cells that hadnot been frozen were analyzed for mitochondrial integrity at eachpassage for 7 passages (over the course of one month of subculturefollowing thawing and plating). The mitochondrial enzyme cytochrome Coxidase showed no difference in activity between frozen and unfrozencardiomyocytes at each passage. Our finding that primary humancardiomyocytes can be stored frozen and later revived allows their“lifetime” to be extended, thus enhancing the usefulness of such cells.

9(f). Endothelial Cells: Human vascular endothelial cells were isolatedas described in section 8 from saphenous vein and aorta obtained frompatients undergoing coronary bypass surgery.

9(g). Smooth Muscle Cells: Human smooth muscle cells were isolated fromsaphenous vein after endothelial cell isolation. After endothelial cellswere collected from veins as described in Section 8, the tissue wasminced and incubated in 10 ml phosphate buffered saline containing 0.2%trypsin, 0.1% collagenase, and 0.02% glucose for 30 minutes at 37° C.The smooth muscle cells were isolated by repetitive pipetting of thedigested tissue. The cells in the supernatant were transferred into atube containing 20 ml of cell culture medium (199 medium containing 20%fetal bovine serum, 100 units/ml penicillin and 100 ug/ml streptomycin),centrifuged at 600×g for 5 minutes at room temperature, resuspended incell culture medium, and cultured.

9(h). Human Fibroblasts: Human fibroblasts were isolated from skinbiopsies as described in Section 6.

10. Rat Cell Purification: Isolated rat cardiomyocytes, smooth musclecells, vascular endothelial cells were purified by a preplatingtechnique (Simpson et al., Circulation Research 51:787-801, 1982) whichtakes advantage of the finding that these cells require a longer time toattach to a cell culture dish than do fibroblast cells. Freshly isolatedcells were plated on dishes and cultured for 2 hours at 37° C., afterwhich the culture medium containing the suspended cells (minus thefibroblasts) was transferred into another dish for further culturing.

The other technique used for culture purification was the clonaldilution technique. When the cells are seeded at low density forculturing, viable cells form individual colonies. Any cell not of thedesired type that is adjacent to a colony of interest is killed with asterile needle. Desired colonies are then collected using a sterilePasteur pipette, and transferred to new culture dishes for culturing andpassaging.

Human cardiomyocytes were used without a purification step.

11. Cell Identification:

Cardiomyocytes: The purity of primary cultures of cardiomyocyte wasassessed by immunofluorescent staining for cardiac myosin heavy chain(Rougier Bio-Tech Ltd. Quebec) (Li R-K, et al., Cardiovascular Research32:362-73, 1996). The cultured cells were fixed with methanol at −20° C.for 15 minutes, washed with phosphate buffered saline, incubated with amonoclonal antibody against cardiac myosin heavy chain for 45 minutes at37° C., washed three times with phosphate buffered saline for 5 minuteseach at room temperature, and then under humid and dark conditionsincubated with rabbit anti-mouse IgG conjugated with fluoresceinisothiocyanate for 45 minutes at 37° C. The cells were washed withphosphate buffered saline, mounted and photographed using a light and UVmicroscope. The purity of cardiomyocyte cultures was determined bycounting the percentage of stained cells in 8 random fields/dish. Eightdishes of cardiomyocyte cultures were used in each cardiomyocytepreparation.

Smooth Muscle Cells: The purity of smooth muscle cell cultures wasdetermined by immunofluorescent staining for α-smooth muscle cell actin(Sigma) as described in the previous paragraph.

Vascular Endothelial Cells: The purity of endothelial cell cultures wasdetermined by immunofluorescent staining for factor VIII as described inthe cardiomyocyte section above.

Fibroblasts: The purity of fibroblast cell cultures was determined byexamining cell morphology with a microscope.

B) Cell Transplantation

Subject animals were grouped into three categories: sham, control andtransplantation. The criteria for such grouping was as follows: GroupSurgical exposure Scar generation Cell transplantation Sham X Control XX Transplantation X X X

1. Surgical Exposure: Sprague-Dawley rats (500 gram) were anesthetizedwith ketamine (22 mg/kg body weight, intramuscular) followed by anintraperitoneal injection of pentobarbital (30 mg/kg body weight). Onceanaesthetized, rats were intubated and positive pressure ventilation wasmaintained with a Harvard ventilator (Model 683, USA). The respiratoryrate was set at 60 cycles/minute with a tidal volume of 2 ml. Animalswere ventilated with oxygen-supplemented room air. The heart was exposedthrough a 2-cm left lateral thoracotomy. The muscle layer and skinincision were surgically closed with 5-0 vicryl sutures.

2. Myocardial Injury and Myocardial Scar Generation: The hearts of theadult rats were exposed as described in the previous paragraph. A5-mm-diameter metal probe cooled to −190° C. for 2 minutes was appliedto the left ventricular free wall of the heart for 20 seconds. Thisprocedure was repeated 8 times. The muscle layer and skin incision werethen surgically closed with 5-0 vicryl sutures.

The animals recovered from surgery in a warm environment, were monitoredfor 4 hours postoperatively and then given Penlog XL (benzathinepenicillin G 150,000 U/ml and procaine penicillin G 150,000 U/ml)intramuscularly (0.25 ml/rat) every three days, and buprenorphine(0.01-0.65 mg/kg body weight) subcutaneously 8-12 hourly for the first48 hours following surgery.

3. Transfection of the Cultured Cardiomyocytes: Freshly isolated orcultured cells were transfected by calcium phosphate coprecipitation(Shi Q-W, et al., Journal of Molecular and Cell Cardiology 24:1221-1229,1992) with a plasmid containing the β-galactosidase gene. Plasmid DNA(20 ug) dissolved in 450 μl of sterile water and 50 μl of 2.5 M CaCl₂was slowly added to 500 μl of aerated 2× HEPES-buffered saline (0.284 MNaCl, 0.050 M HEPES acid, and 1.48 mM Na₂ HPO₄, pH 7.05). After 20minutes at room temperature, the solution was added to a cardiomyocytesuspension (1.0×10⁶ cells/6 ml of culture medium). In the control groupsthe same procedure was performed either without plasmid DNA or withpREP4 (a plasmid that lacks a β-galactosidase gene; Invitrogen, USA;plasmid control). The cells were cultured at 37° C., 5% CO₂ and 95% airfor 24 hours.

To determine the efficiency of cell transfection, cells cultured foreither 24 hours or 4 weeks were washed three times with phosphatebuffered saline, fixed in 2% formaldehyde and 2% glutaraldehyde inphosphate buffer (0.15 M NaCl and 0.015 M NaH₂PO₄, pH 7.2) at 4° C. for5 minutes, washed with phosphate buffer containing 2.0 mM MgCl₂, andstained overnight at 37° C. in a solution containing 1 mg/ml5-bromo-4-chloro-3-indolyl-beta-galactopyranoside (X-gal), 5 mMK₃Fe(CN)₆, 5 mM K₄Fe(CN)₆3H₂O, 0.2 mM MgCl₂ in phosphate buffer (pH7.2). The stained and non-stained cells in 8 random fields/dish (6dishes from 6 preparations) were counted under the microscope todetermine the percentage of cells containing β-galactosidase activity.Two dishes from each transplant experiment were used for a celltransfection efficiency study (N (number of animals)=6).

4. Cell Preparation and Transplantation: Cultured cells were washedthree times with phosphate buffered saline to remove dead cells and thendetached from the cell culture dish and each other with 0.05% trypsin inphosphate buffered saline for 3 minutes. After adding 10 ml of culturedmedium, the cell suspension was centrifuged at 580×g for 3 minutes. Thecell pellet was resuspended in culture medium at a concentration of 4×10⁶ cells/ml culture medium. The volume of cell suspension was 0.25 ml foreach cell transplantation.

On day 14 or 30 following myocardial scar generation (Section 2), theanimals (which now had mature transmural scars in their ventricles) wereanesthetized as described in Section 1. The scar tissue in the heart wasexposed through a midline sternotomy. The cell suspension (0.25 ml) wasinjected into the scar tissue of the animals of the transplant groupusing a tuberculin syringe. Control animals were similarly injected with0.25 ml of culture medium (no cells). The rats in the sham groupunderwent the surgical procedure without injection. Cryo-precipitate(fibrin glue) was placed on the injection sites to prevent leakage ofthe injected cells. The chest was closed with 5-0 vicryl sutures.Antibiotics and analgesia were given as described in Section 1.Cyclosporin A, at a dose of 5 mg/kg body weight/day, was administeredsubcutaneously into animals of all three groups. The rats were housed incages with filter tops.

C) Measurement of Heart Function

Eight weeks after myocardial injury, the heart function of sham, controland transplanted animals was measured using a Langendorff preparation(Stephen S E, et al., Annals of Thoracic Surgery 59:1127-1133, 1995).The rats were anesthetized and heparin (200 units) was administeredintravenously. The hearts were quickly isolated from rats and perfusedin a Langendorff apparatus with filtered Krebs Heinseleit buffer (mmol/LNaCl 118, KCl 4.7, KH₂PO₄ 1.2, CaCl₂ 2.5, MgSO₄ 1.2, NaHCO₃ 25, glucose11; pH 7.4) equilibrated with a 5% CO₂ and 95% O₂. A latex balloon waspassed into the left ventricle through the mitral valve and connected toa pressure transducer (Model p10EZ, Viggo-Spectramed, Calif.) andtransducer amplifier and differentiator amplifier (Model 11-G4113-01,Gould Instrument System Inc., Ohio). After 30 minutes of stabilization,coronary flow in the heart was measured in triplicate by timedcollection in the emptying beating state. The balloon size was increasedby addition of saline in 0.02 ml increments from 0.04 to 0.8 ml, or thevolume at which end diastolic pressure reached to 30 mm Hg, or whichevercame first. The systolic and diastolic pressures were recorded at eachballoon volume and developed pressure was calculated as the differencebetween the systolic and diastolic pressures.

Hearts were weighed and their sizes were measured by water displacement.

D) Measurement of Left Ventricular Remodeling

The epicardial and endocardial surface areas of the normal and scartissue in the left ventricular free wall (LVFW) were measured by thetechniques of Pfeffer et al (Pfeffer J M, et al., American Journal ofPhysiology 260:H1406-14, 1991) and Jugdutt and Khan (Jugdutt B I, etal., Circulation 89:2297-307, 1994). Briefly, the hearts were fixed indistension (30 mm Hg) with 10% phosphate-buffered formalin solution andthen cut into 3 mm thick sections. For each section, the area of normaltissue, scar tissue, and transplanted tissue in the left ventricularfree wall were traced onto a transparency and quantified usingcomputerized planimetry (Jandal Scientific Sigma Scan, USA) as describedby Wu et al. (Wu T W, et al., Cardiovascular Research 27:736-39,1993).The lengths of left ventricular free wall and scar tissue on both theendocardial and epicardial surfaces of each section were measured. Thesurface areas of the epicardial and endocardial scar tissue and leftventricular free wall were measured as:(endocardial length+epicardial length)×section thickness (3 mm).The surface area percentage of scar tissue in the left ventricular freewall (LVFW) was calculated as:$\frac{\left( {{{epicardial}\quad{scar}\quad{size}} + {{endocardial}\quad{scar}\quad{size}}} \right)}{\left( {{{endocardial}\quad{LVFW1}} + {{epicardial}\quad{LVFW}}} \right)} \times 100$

To calculate the percentage of the surface area in the scar tissueoccupied by the transplanted tissue, the following equation was used:$\frac{\begin{matrix}{\left( {{cardiac}\quad{tissue}\quad{length}\quad{in}\quad{the}\quad{scar}\quad{tissue}\quad{of}\quad{each}\quad{section}} \right) \times} \\\left( {{section}\quad{thickness}\quad\left( {3\quad{mm}} \right)} \right)\end{matrix}}{\left( {{total}\quad{scar}\quad{area}} \right)} \times 100$E) Histology and Electron Microscopy of Transplanted Cells

On day 30 or 45 post-transplantation, the animals were anesthetized asin Example 1 and transplant, control, and sham hearts were exposedthrough a midline sternotomy and quickly harvested. The animals wereeuthanised by exsanguination under general anesthesia.

To localize the tissue formed by transplanted cells, the transplantedand control myocardial scar tissues were fixed in 5% glacial acetic acidin methanol. The tissue was embedded in paraffin and cut into 10 μmthick sections. After removal of the paraffin by immersing the sectionsfor 3 minutes in xylene and then in 100, 95, 90, 85, and 70% ethanol for3 minutes each, the samples were stained with haematoxylin and eosin asdescribed by the manufacturer (Sigma Diagnostics, St. Louis, Mo.), andphotographed.

To stain for β-galactosidase activity in the transplantedcardiomyocytes, the heart sections were fixed at 4° C. for 12 hours in2% formaldehyde and 2% glutaraldehyde in phosphate buffer (0.15 M NaCland 0.015 M NaH₂PO₄, pH 7.2). The transplanted cardiomyocytes werelocalized by staining for β-galactosidase activity as described earlierin Example 2. The stained tissue was embedded in paraffin and cut into10 μm thick sections that were stained with haematoxylin and eosin asdescribed in the last paragraph.

To identify cultured ventricular and atrial cardiomyocyte transplants,the transplanted heart tissue was immunofluorescently stained forcardiac myosin heavy chain. Briefly, tissue sections were washed threetimes with phosphate buffered saline and fixed with 2 ml 100% coldmethanol at −20° C. for 15 minutes. After washing three times withphosphate buffered saline and drying by draining, the tissue sectionswere exposed for 45 minutes at 37° C. to monoclonal antibodies againstcardiac myosin heavy chain (Rougier Bio-Tech, Montreal, Canada), diluted1:20 with saline. Control tissues were incubated under the sameconditions with phosphate buffered saline. The tissues were washed threetimes with phosphate buffered saline for 15 minutes at room temperaturewith gentle shaking, after which the secondary antibody, rabbitanti-mouse IgG conjugated with fluorescein isothiocyanate at aconcentration of 1:32 dilution with phosphate buffered saline, wasadded. The tissues were incubated with the second antibody under darkand humid conditions for 45 minutes at 37 ° C. After washing withphosphate buffered saline, the cells in the transplant control tissueswere visualized under ultraviolet light using an epi-microscope with ablue filter.

The smooth muscle cell transplants were identified by stainingimmuno-fluorescently using a monoclonal antibody for a-smooth muscleactin as the primary antibody. Endothelial cell transplants wereidentified by immunofluorescent staining for factor VIII, as describedin the next section.

Fibroblast transplants were identified by the presence of a localizedimmunorejection within the ventricular scar.

F) Histological and Electron Microscopy Studies of Angiogenesis in theGraft

For immunocytochemical staining of factor VIII-related antigen in thevascular endothelial cells, tissue sections processed as in Section Eabove were incubated with xylene twice for 5 minutes each, 100% ethanoltwice for 2 minutes each and then with 70% ethanol twice for 1 minuteeach. The sections were incubated with rabbit IgG against factorVIII-related antigen (Dimension Lab. Inc., Ontario). The control sampleswere incubated with phosphate buffered saline under the same conditions.The test and control samples were incubated with goat anti-rabbit IgGconjugated with peroxidase. After washing the samples three times withphosphate buffered saline following secondary antibody staining, thesamples were immersed in diaminobenzidine-H₂O₂ (2 mg/mldiaminobenzidine, 0.03% H₂O₂ in 0.02 M phosphate buffer) solution for 15minutes. The samples were washed with phosphate buffered saline. Thestained vascular endothelial cells in the grafts (N=17) and controlgroups (N=14) were counted using a light microscope at 200×magnification. The result was expressed as number of blood vessels/fieldarea (0.8 mm²).

For electron microscopy studies, hearts were fixed in 1% glutaraldehydein phosphate buffer, postfixed with 1% osmium tetroxide, dehydrated ingraded ethanol (50, 70, 90 and 100%), polymerized in propylene oxide at60° C. overnight, sectioned, and examined using a JEOL 1200 TEM electronmicroscope (Li R-K, et al., Cardiovascular Research 32:362-73, 1996).

G) Cell Grafts

Cultured cardiomyocytes, smooth muscle cells, endothelial cells, and/orfibroblasts were seeded onto biological mesh, such as a collagenmembrane, and on non-biological membranes, such as non-degradablemembranes (Dacron) or degradable membranes (polyglycolic acid polymers),and the mesh and cells were cultured in cell culture medium. Seven daysafter culture, the cell-containing mesh was fixed in 2% formaldehyde and2% glutaraldehyde in phosphate buffer (0.15 M NaCl and 0.015 M NaH₂PO₄,pH 7.2) at 4° C. for 12 hours, embedded in paraffin, and cut into 10 μmthick sections, which were stained with haematoxylin and eosin asdescribed in Section E, and photographed.

H) Cell Gluing Technique for Cell Transplantation

To be successful in preventing failure of the infarcted heart,sufficient cardiomyocytes must be implanted into the infarctedmyocardium. Although this can be done by multiple syringe injections,injecting the cells, unfortunately, limits the number of cells which canbe transplanted into myocardial scar tissue. We have investigatedanother technique to apply a large number of cells onto the infarctedmyocardium. Thrombin and cryoprecipitate (fibrin glue), which arederived from human blood, clot rapidly. Our in vitro results showedsurvival and contraction of cardiomyocytes in fibrin clots. We used thisfibrin glue for cell transplantation.

1. Gluing Injection Site to Prevent Transplanted Cell Leakage: Thebiological glue is applied onto the injection site while the injectionneedle is still in place. The needle is withdrawn after the glue clots.In such manner, leakage of transplanted cells, as discussed previously,is prevented.

2. Gluing the Cells onto the Myocardial Scar Tissue to Prevent InjectionDamage: We removed the epicardium over the scar and damaged myocardium.The transplanted cells were suspended in thrombin, and the thrombin/cellsuspension was applied onto the myocardial scar tissue withcryoprecipitate (fibrin glue). The glue caused the cell suspension toadhere to the surface of both the scarred and normal myocardium, afterwhich the pericardium was glued on top of the fibrin clot. Cell loss wasprevented. We found that glued cardiomyocytes can survive on themyocardial scar tissue and permit a large number of cells to betransplanted. This technique improved heart function better than onlyinjecting the cells. The cardiomyocytes glued directly onto themyocardium without epicardium can connect with host cardiomyocytes andcardiomyocytes to contract synchronously with the host myocardium.

3. Gluing the Cellular Mesh onto the Myocardial Scar Tissue to PreventIts Expansion: Cell grafts comprising biological or non-biological meshhaving cells suspended thereon (as described in Example 7) were gluedonto the scar tissue. The pericardium was in turn glued on top of themesh.

I) Data Analysis

Data are expressed as the mean+standard error. The Statistical AnalysisSystem software was used for all analysis (SAS Institute, Cary, N.C.).Comparisons of continuous variables between more than two groups wereperformed by a one-way analysis of variance. If the F ratio wassignificant from the analysis of variance, a Duncan's multiple-range ttest was employed to specify differences between the groups. Alpha forthese analyses was set at p<0.05.

Function data were evaluated for the sham, control and transplant groupsby an analysis of covariance using intracavitary balloon volume as thecovariate and systolic, diastolic and developed pressure as dependentvariables. Main effects were group, volume, and the interaction betweengroup x volume. If there was an overall difference in the analysis ofcovariance, multiple pair-wise comparisons were performed to specifywhich groups were different. Because there were multiple pair-wisecomparisons, a Bonferroni correction was performed and the criticalalpha level was set at 0.01 for the analysis of covariance.

J) Use of Growth Factors in Treating Idiopathic HypertrophicCardiomyopathy (HCM)

Idiopathic hypertrophic cardiomyopathy. (HCM) is a primary cardiacabnormality characterized by regional asymmetrical myocardialhypertrophy. The hypertrophic myocardium can result in obstruction ofleft ventricular ejection as well as systolic and diastolic dysfunctionand myocardial ischemia. Symptoms unresponsive to medical therapy cannecessitate surgery.

HCM is described for the most part as a heterogeneous disease of thesarcomeres. At least 34 missense mutations have been described in theβ-myosin heavy chain gene, and 7 mutations in candidate loci also exist.However, family studies suggest that the autosomal dominant traitaccounts for only 50% of HCM patients. The remaining HCM patients showno familial transmission and the disease occurs sporadically. Myocardialcalcium kinetics and sympathetic stimulation have been studied becauseof diastolic functional abnormalities. However, none of these findingsexplains the regional myocardial hypertrophy (cardiomyocyte hypertrophyand over production of extracellular matrix proteins) observed in mostHCM patients. The etiology of this disease remains unknown. It isthought that growth factors may play an important role in cardiomyocyteproliferation, cell hypertrophy and the overproduction of extracellularmatrix.

To investigate the involvement of growth factors in myocardialhypertrophy in HCM patients, we evaluated gene expression and cellularlocalization of transforming growth factor β1 (TGFβ1), insulin-likegrowth factors (IGF-I, -II), and platelet-derived factor-B (PDGF-B) inventricular biopsies obtained from patients with HCM (N=8), aorticstenosis (AS, N=8), stable angina (SA, N=8), and explanted hearts withischemic cardiomyopathy (TM, N=7).

Methods: Levels of TGFβ1, IGF-I, IGF-II and PDGF-B transcripts werequantitated using multiplex RT-PCR. Glyceraldehyde 3-phosphatedehydrogenase (G3PDH) was used as an internal standard. Antibodiesagainst TGFβ1 and IGF-I were used to localize their peptides within themyocardium. Antisense and sense (control) cRNA probes for TGFβ1 andIGF-I, labeled with digoxigenin, were used to localize the growth factortranscripts by in situ hybridization.

Results: MRNA levels (densitometric ratio of GF/G3PDH) of TGFβ1 andIGF-I in HCM myocardium (0.75±0.05, 0.85±0.15, mean±1SE) weresignificantly (p<0.01 for all groups) elevated in comparison to non-HCMmyocardium (AS: 0.38±0.07, 0.29±0.06; SA: 0.32±0.04, 0.18±0.05; TM:0.25±0.03, 0.15±0.03). mRNA level of TGFβ1 and IGF-I in the hypertrophicAS myocardium were greater (p=0.02, p=0.05) than those in the explantedmyocardium (TM). Immunohistochemical and in situ hybridization studiesshowed increased expression of TGFβ1 and IGF-I in the HCMcardiomyocytes.

Conclusion: Gene expression of TGFβ1 and IGF-I was enhanced inidiopathic hypertrophy and may be associated with its development.

Results

1 . Ventricular and Atrial Cardiomyocytes: Fetal and adult mammalianventricular cardiomyocytes and adult mammalian atrial cardiomyocyteswere isolated using enzymatic digestion as described above in Sections1-3, Example I. After purification, the purity of the culturedventricular and atrial cardiomyocytes was greater than 94% (N=8) asdetermined by the percentage of cells that stained for cardiac myosinheavy chain. The cardiomyocytes grew in vitro, connected with each otherand formed a tissue pattern after six days of culture. The fetalcardiac-like tissue contracted regularly and spontaneously in culture.The adult cardiomyocytes did not contract in culture.

2. Smooth Muscle Cells: Fetal and adult smooth muscle cells weresuccessfully cultured. The cells proliferated in the culture. Thecultured cells were stained strongly with antibodies against α-smoothmuscle actin.

3. Vascular Endothelial Cells: Endothelial cells were isolated fromblood vessel and cultured. Staining with antibody against factor VIIIshowed that more than 95% cells in the culture dish were endothelialcells.

4. Fibroblasts: Fetal and adult skin fibroblasts were isolated andcultured. The cells proliferated in the culture condition. When theculture reached confluency, the cells formed a typical fibroblastpattern: spindle-shaped cells in a wave pattern.

5. Cell Transfection: Twenty-four hours after transfection of freshlyisolated cells, the percentage of the transfected cells withβ-galactosidase was 18.2±5.2% (N=6). No cells stained positively in thecontrol groups with plasmid pREP4 (N=6) and without a plasmid (N=6).After culturing for 4 weeks, 5.4±3.1% (N=6) of the transfected cellsstained positively for β-galactosidase activity.

6. Myocardial Scar Tissue: Immediately after myocardial injury, 25±3% ofthe left ventricular free wall (LVFW) was transmurally damaged. Thecardiomyocytes were fragmented. At one week, most of the necrosedcardiomyocytes were gone and a predominantly mononuclear inflammatoryinfiltrate was present in the affected area. At two weeks theinflammatory infiltrate had almost disappeared and fibroblasts andcollagen deposition were evident. At four and eight weeks, the scar wascomposed of fibrotic tissue. The tissue was less cellular andlymphocytes were not observed. No cardiac muscle cells were observed inany scar tissue.

The myocardial scar of the left ventricle increased in size over the 8week study period in the control hearts. Although the scar sizes at 1and 2 weeks (13±6% and 21±4% of left ventricular free wall) were notstatistically different, the scar size after 4 weeks (39±5% of leftventricular free wall) was greater (p<0.01). At 8 weeks there was afurther increase (p<0.01) in scar size (55±3% of left ventricular freewall).

7. Optimal Time for Cell Transplantation: The fetal rat cardiomyocytestransplanted into myocardial tissue immediately after myocardial damagedid not survive in the affected area. The scar area (53±5%) oftransplanted hearts was similar to that of the control group (55±3% ofthe left ventricular free wall). Cardiomyocytes transplanted at 2 weeksafter myocardial damage formed cardiac tissue that occupied 34% of thetotal scar area (11±3% of the left ventricular free wall). Similarly,cardiomyocytes transplanted at 4 weeks occupied 32% of total scar area(14±4% of left ventricular free wall). The scar sizes in the hearts thatreceived transplants 2 weeks and 4 weeks after myocardial damage weresmaller (p<0.01) than the scar size of the control hearts. The scar sizeof the hearts that received transplants 2 weeks after myocardial damagewas smaller (p<0.01) than that of the hearts that received transplants 4weeks after myocardial damage.

8. Transplanted Cells in Myocardial Scar Tissue: Cells were transplantedinto transmural scars two and four weeks after scar cryo-induction. Sixweeks or four weeks after ventricular cardiomyocyte transplantation(i.e., eight weeks after scar induction in all animals), only fetalcardiomyocyte tissue (N=17) had formed within the myocardial scartissue. The cells were connected to each other and formed a cardiactissue pattern. The tissue of all 3 animals, transplanted withcardiomyocytes transfected with the β-galactosidase gene, containedβ-galactosidase activity. The transplanted cardiomyocytes containedsarcomeres and were connected by junctions composed of desmosomes andfascia adherens, which were not present in the cardiomyocytesimmediately prior to transplantation. Lymphocytic infiltrationsurrounded the cardiac tissue formed by transplanted fetalcardiomyocytes. In the control animals (N=14), cardiac tissue,lymphocytic infiltration, and β-galactosidase activity were not observedin the scar. The eight-week scar was less cellular than the four-weekold scar tissue.

Adult rat atrial cardiomyocytes were autotransplanted into the scartissue of the same rat. At 6 weeks after transplantation, thetransplanted cells were detected in the myocardial scar tissue bystaining with an antibody against cardiac myosin heavy chain. There wasno lymphocytic infiltration. No cardiac tissue was found in the scartissue of control rats.

At 6 weeks after transplantation, scars transplanted with either allo-or auto-smooth muscle cells contained smooth muscle tissue that stainedpositively for α-smooth muscle actin. The allotransplants wereinfiltrated with lymphocytes, indicative of immuno-rejection. Incontrast, there was no lymphocytic infiltrate or signs ofimmunorejection in the auto-smooth muscle cell transplant.

FIG. 1 shows the improved cardiac function of scarred heartstransplanted with smooth muscle cells. Hearts were cryo-injured, afterwhich the mature scar tissue was transplanted with allogenic smoothmuscle cells. Function measurements were made four weeks aftertransplantation. The graph shows developed balloon pressures of sham(top line; no cryo-injury, no transplantation), control (bottom line;cryo-injury, no transplantation), and transplanted (middle line) heartswith increasing balloon volumes. Developed pressures of transplantedhearts were significantly higher than control hearts (p=0.0001), butlower than that sham of sham hearts (0=0.0001).

At 6 weeks after transplantation, the transplanted fibroblastsproliferated in the myocardial scar tissue. The cells secretedextracellular matrix, which increases the thickness of the scar tissue.The transplanted cells also stimulated angiogenesis (see below), whichsurvived damaged myocardial tissue in the scar tissue. There were nocardiac muscle cells in the scar tissue in control animals. The scartissue at the left ventricular free wall of the control hearts dilatedduring systole, whereas scar tissue containing transplanted cells wasimmobile. Although the surface area of transplanted scar tissue(22.9±6.2 mm²) was similar to that of control scar tissue (23.8±6.5mm²), scar thickness (1.9±0.9 mm, N=12) in hearts containingtransplanted cells was twice (p<0.01) that of the control hearts(1.0±0.4 mm, N=10). Consistent With the above findings, the leftventricular volume of the transplanted hearts was 275±48 mm³ (N=12)which was less (p<0.01) than the 360±94 mm³ volume of the control hearts(N=10).

Although a detailed observation was not made of transplanted endothelialcells, staining with antibody against factor VIII revealed that morecells stained positively in scar tissue transplanted with endothelialcells than in the control scar tissue.

9. Angiogenesis in Myocardial Scar Tissue after Cell Transplantation: At4 to 6 weeks after transplantation, examination by histology andelectron microscopy showed that angiogenesis occurred in thetransplanted fetal ventricular cardiomyocyte transplant. Significantlymore arterioles and venules were found (p<0.01) in the cardiomyocytegrafts (1.2±0.6 vessels/0.8 mm², N=14) than in the control myocardialscar tissue (0.1±0.1 vessels/0.8 mm², N=14).

Similarly, angiogenesis occurred in the atrial cell, endothelial cell,smooth muscle cell and fibroblast cell transplants.

10. Transplanted Cells Limited Scar Expansion: At 4 to 6 weeks aftertransplantation of the cells, the heart rate and coronary flow did notdiffer among the sham, control and transplanted animals. The controlmyocardial wall at the site of injury had thinned and consisted offibrous tissue and a few blood vessels. No cardiac muscle or lymphocyteswere present. The control and transplanted hearts were larger (p<0.01)than the sham (undamaged) hearts. At 4 weeks after cryo-injury,36.4±4.4% (mean±1 SE, N=5) of the left ventricular free wall in thepretransplant animals was replaced with a transmural scar. At 8 weeks,the scar tissue expanded (p<0.01) in the control group to 54.6±2.9%(N=5) of the free wall for the fetal cardiomyocytes. The scar tissue inthe transplanted animals was 43.4±1.8% (N=5) of the left ventricularfree wall. This did not significantly differ from the pretransplantedanimals at 4 weeks after cryo-injury and was less (p<0.05) than thecontrol hearts at 8-weeks after cryo-injury. The transplantedcardiomyocytes formed cardiac tissue which occupied 36.5±3.5% (N=5) ofthe scar tissue. The transplanted tissue visibly contracted. We wereunsuccessful in measuring its contractility because of the contractionsof the heart. After removing the hearts and separating the scar area,the transplanted region continued to contract when stimulated.

Similarly the atrial cell, smooth muscle cell, and fibroblasttransplants limited scar expansion.

11. Improvement of Heart Function by Transplanted Cells: Ventricularfunction of hearts into which cells were transplanted immediately aftermyocardial injury was similar to that of control hearts. Analysis ofcovariance demonstrated no interaction between balloon volume andtreatment group for developed pressures. Ventricular function of thetransplanted and the control hearts when the cardiomyocytes weretransplanted at 2 weeks after myocardial injury was different because ananalysis of covariance demonstrated a significant (p<0.05) interactionbetween balloon volume and treatment group for developed pressures. Thetransplanted hearts had better (p<0.05) function than the controlhearts. Similarly cells transplanted at 4 weeks after myocardialnecrosis improved (p<0.05) myocardial function. The hearts transplantedat 2 weeks had higher (p<0.05, p<0.05, p<0.05) developed pressures atballoon volumes 0.1, 0.2, and 0.3 ml than hearts transplanted at 4weeks.

In the measurements of ventricular function in the sham-operated, thetransplanted and the control hearts, an analysis of covariancedemonstrated a significant (p<0.05) interaction between balloon volumeand treatment group for systolic, diastolic and developed pressures.Pairwise comparisons demonstrated a significant (p<0.05) depression insystolic and developed pressures in control animals compared to thesham-operated normal hearts. The transplanted hearts had better (p<0.05)function than the control hearts although both systolic pressure anddeveloped pressure were lower (p<0.05) than the sham-operated normalhearts. Diastolic pressures were significantly lower in both thecryo-injured controls and the transplanted hearts than sham-operatednormal hearts at higher balloon volumes due to the marked dilatationresulting from myocardial scar expansion.

12. Tissue Engineered Grafts: Cardiomyocytes, smooth muscle cells,fibroblasts, and endothelial cells were observed in the mesh of thegrafts. The tissue that formed in the mesh stained strongly withhaematoxylin and eosin.

Summary

From the above results, it can be seen that we have successfullytransplanted muscle and non-muscle cells in cardiac scar tissue. Thesecells formed viable tissue, altered structure of the scar, improvedheart function, stimulated angiogenesis, and expressed a gene foreign tothe scar. From these results, the following conclusions are drawn:

1. Cultured adult atrial cardiomyocytes could be successfullytransplanted into the scar. The atrial tissue could be digested and theatrial cells immediately transplanted or the atrial tissue could bedigested, cultured and passaged up to five times and then transplanted.Auto- and allo-transplantation of cultured adult atrial cardiomyocytesformed tissue within the scar. Heart function improved. Angiogenesisoccurred. No immunorejection occurred with auto-transplantation of theadult atrial cardiomyocytes.

2. Smooth muscle cells can be successfully auto- or allo-transplantedinto the scar. Smooth muscle tissue formed within the scar. Noimmunorejection occurred with the auto-transplantation. Angiogenesisoccurred. Heart function improved. The cells can be freshly isolated, orcultured and passaged before transplanting.

3. Fibroblasts can be successfully transplanted into scar tissue. Scarthickness increased. Heart function improved. Angiogenesis occurred.Host cardiomyocytes survived in the scar. The cells can be freshlyisolated, or cultured and passaged before transplanting.

4. The addition of cryo-precipitate to the injection site preventedleakage of the transplanted cells from the scar.

5. A plasmid containing a foreign gene to the scar tissue and hearttissue was transfected into the cultured cells to be transplanted. Thecells were successfully transplanted into the scar tissue and expressedthe foreign gene.

6. Cardiomyocytes, smooth muscle cells, skeletal muscle cells,fibroblasts, and endothelial cells can be successfully transplanted intofibrous membranes and non-degradable or biodegradable polymers to formtissue. The product of such a process would be a patch which can havevarious clinical and therapeutic uses. Such membranes may be made fromDacron or biodegradable sheets such as polyglycolic acid polymers withor without polylactic acid polymers. Such a patch can be associated witha pacemaker and be implanted close to a cardiac defect thereby providinga means of paced cardiomyoplasty.

7. Cell combinations could be successfully transplanted to form tissuewithin a scar to improve function, to stimulate angiogenesis and to formtissue.

8. The optimal time for transplantation is immediately after the acuteinflammatory response to the myocardial injury has disappeared.

9. Adult mammalian atrial and ventricular cardiomyocytes can besuccessfully isolated from human myocardium, cultured, passaged andstored using the cell culture procedure described for humancardiomyocytes described in the Human Cell Isolation, Culturing andStorage section. Human cardiomyocytes can be prepared for long termstorage in liquid nitrogen and thawed for culturing as described. Suchcultured cells can then be used in forming grafts as discussed above.

10. The biological glue technique used in the cell transplantationprocedure increased the number of cells transplanted in myocardial scartissue. This invention enhanced the transplant success rate andmaximized the improvement of heart function.

Co-transplantation of growth factors such as IGF-I, IGF-Il, TGF-β1 andPDGF-B increases the survival of transplanted cells, inducestransplanted muscle hypertrophy, and stimulates angiogenesis. Based onthese findings, the use of other growth factors such as fibroblastgrowth factor and vascular endothelial growth factor are also possible.Such growth factors can be co-transplanted either alone or incombination. These techniques can increase transplanted muscle size andsurvival in myocardial scar tissue and damaged myocardial tissue.

11. Cultured cells can be employed to restore regional cardiac functionand blood flow to regions in the heart damaged by acquired diseaseprocesses or absent because of congenital defects. During reconstructivesurgery, the defective portion of the heart is removed and replaced withinert materials to secure a water-tight seal. Attaching a contractingtissue of myocytes to the reconstruction patch will permit the return offunction to the destroyed region. Implantation of a collection of cellscould restore function and improve the quality of life of the patientsundergoing reconstructive surgery.

12. Most acquired or congenital cardiac defects are closed with inertmaterials intended to provide a water-tight seal. Instead of an inertmaterial, a graft consisting of a biodegradable or non-biodegradablescaffolding supporting cultured cells can be employed to close suchcardiac defects. The graft would employ myocytes enhanced with genes toincrease perfusion and contractility (by increasing the size and numberof myocytes). In addition, the endothelial cells can be applied to theblood surface of the graft to prevent intravascular clot formation inthe heart. The endothelial cells could be genetically engineered so thatthey produce proteins that prevent clot formation on the blood surfaces.The cellular grafts will permit closure of acquired and congenitalcardiac defects with functioning tissue which may improve heartfunction.

13. Cardiac surgeons frequently remove segments of the heart which havebeen damaged or are defective due to congenital abnormalities. Acellular graft permits an opportunity to restore function to regionswhich are usually closed with inert materials. The graft may consist ofmyocytes grown on biodegradable scaffolding. Endothelial cells can begrown on the blood interface layer. The cells will create a functioninggraft which will replace the region removed at the time of surgery. Thefunctioning myocardial graft could restore function for patients whohave suffered a myocardial infarction and require removal of thedestroyed regions. The functioning graft could re-establish systematicand/or pulmonary circulation for patients with extensive congenitaldefects undergoing reconstructive surgery. A functioning myocardialgraft offers the promise of restoring an improved quality of life todisabled individuals.

Effect of Donor Age on Contractility of Transplanted Rat Cardiomyocytes

The inability of heart muscle to regenerate following myocardialnecrosis is an important clinical problem. Our previous research hasshown survival and contractility of transplanted fetal ratcardiomyocytes (CMs) in adult rat skeletal muscle. The present studyfocuses on the effect of donor age on contractility of transplanted CMs.

Methods:

CMs were isolated from 18-day gestation, 5-, 22-, 32-, and 62-day oldSprague-Dawley rat hearts and cultured for 1 day. 2 to 5×10⁶ cells insaline were injected into the skeletal muscle of one adult rat leg. Theother leg (control) was injected with saline alone. Cell viability andfunction were assessed visually and by ultrasound andelectrocardiography (ECG).

Results:

In the groups with transplanted fetal (n=12) and neonatal (n=4) CMs,rhythmic contractions (73±12 beats/min (n=l 1) and 43±21 beats/min(n=3)) were found only in the transplanted area. These contractions werenot observed in the control legs. Contractions were not observed atsites transplanted with CMs from 22-, 32-, and 62-day donor animals (n=4for all groups).

Conclusion:

Donor age is important for maintaining contractility of CMs aftertransplantation into skeletal muscle.

Induction of Angiogenesis by Endothelial Cells Transplanted intoMyocardial Transmural Scars did not Improve Cardiac Function inInfarcted Hearts.

We speculated that artificial induction of angiogenesis might improveperfusion, alleviate angina and restore cardiac function in cases whererevascularization does not occur spontaneously after myocardialinfarction (e.g., in scar tissue). The present study evaluated whetherallogenic and syngenic endothelial cells transplanted into myocardialscar tissue induce new blood vessel formation and whether angiogenesis(if any) induced by transplanted cells affects the function of thescarred heart. The effect of transplanted cells upon blood vesselformation in myocardial scar tissue has not been studied previously.

Methods and Results: Transmural scars were produced at the leftventricular free wall of adult rats hearts by cryo-injury. Two weekslater, cultured adult rat aortic endothelial cells (transplanted group)or culture media (control group) were injected into the myocardial scarsof rats immunosuppressed with cyclosporin A (25 mg/kg/day). Transplantedcells were observed at transplanted sites at day 1 and 7, but not at day14 (n=2). At 6 weeks after transplantation, the number of capillaries inthe scars of the transplanted group (3.4±2.0, n=10) was greater (p<0.0l)than that in the control group (1.0±0.6, n=9). Blood flow measured byradioactive ⁵⁷Co labeled microspheres was greater (p<0.01) in thetransplanted group (6.85±2.27%, n=9) than in the control group(3.78±0.99%, n=8). There was no difference in scar size and heartfunction between two groups measured by planimetry and Langendorfpreparation.

Conclusion: Angiogenesis was induced in the scar tissue by endothelialcell transplantation. Since no myocytes were present in the scar,function did not improve. Endothelial cell transplantation in patientswith incomplete infarction may restore perfusion, relieve angina andrestore function.

Methods

Experimental Animals: All procedures performed on animals were approvedby the Animal Care Committee of The Toronto Hospital. Experiments wereperformed according to the “Guide to the Care and Use of ExperimentalAnimals” published by the National Institutes of Health (NIH publication85-23, revised 1985). Sprague-Dawley rats (Charles River Canada Inc,Quebec, Canada) were used for allogenic transplantation. Male rats,weighing 330 to 360 g, were used as recipients and donors of endothelialcells. Sygenic Lewis rats (Male, 250-300 g, Charles River Canada Inc,Quebec, Canada) were used for autologus transplantation.

Cell Isolation and Culture: Endothelial cells from rat aorta wereisolated and cultured as previously described (Mickle et al., J Mol.Cell. Cardiol. 22:1297-1304, 1990). In brief, descending thoracic aorticsegments were obtained from rats under general anesthesia as describedin the next section. The blood vessels were washed with phosphatebuffered saline solution (PBS; composition in mmol/L: NaCl 136.9, KCl2.7, Na₂HPO₄ 8.1, KH₂PO4 1.5, pH 7.3) and connective tissue was removed.The aortic segment was then flushed with enzyme solution (0.1%collagenase and 0.2% trypsin in PBS) and incubated with the enzymesolution for 30 minutes at 37° C. After incubation, endothelial cellswere isolated by flushing the inside of aortic segments with culturemedium (Medium 199 containing 20% fetal bovine serum, 100 U/ml ofpenicillin, 100 ug/ml of streptomycin, and 0.5% heparin salt) three tofive times. The isolated cells were cultured at 37° C. in 5% CO₂ and 95%air in culture medium.

Cell Identification: The purity of endothelial cell cultures wasconfirmed by a immunohistochemical stain using antibody against factorVIII-related antigen as previously described (Mickle et al., J. Mol.Cell. Cardiol. 22:1297-1304, 1990). In brief, the cultured endothelialcells were fixed in methanol for 15 minutes at −20° C. After washingthree times with PBS, the cells were incubated with rabbitimmunoglobulin G against factor VIII-related antigen (DimensionLaboratory, Inc) at 37° C. for 45 minutes. After incubation, the cellswere washed three times (15 minutes per wash) with PBS, then incubatedwith goat anti-rabbit immunoglobulin G conjugated with peroxidase at 37°C. for 45 minutes, washed three times (15 minutes per wash) with PBS,and immersed in diaminobenzidine-H₂O₂ (2 mg/ml diaminobenzidine, 0.03%H₂O₂ in 0.02 ml/l phosphate buffer) solution for 15 minutes. After afinal wash in PBS, the samples were photographed.

Quantitation of VEGF Protein Levels in Cultured Endothelial Cells andSecreted by Cultured Cells: Purified endothelial cells were cultured incell culture medium for 3 days. The supernatants (n=5) were collected,centrifuged at 14,000 g for 10 min. to remove cell debris, and weresaved for protein quantitation. The cultured cells (n=5) were washed 3times with PBS and collected by scraping with cell scraper. Aftercentrifugation at 580 g for 3 min., the cell pellets were resuspended in1 ml of PBS and sonacated for 1 min. The samples were centrifugated at14,000 g for 10 min. The supernatants were collected for proteinquantitation. The protein concentration was measured using the Bio-Radprotein assay (Bio-Rad, Richmond, Calif.). VEGF protein levels inculture medium and endothelial cells were quantitated bychemiluminescent slot blot analysis. Culture medium incubated withoutcells was used as a control. Forty micrograms of protein from culturemedium or from cultured endothelial cells were loaded onto a 0.2 μmnitrocellulose membrane (Schleicher & Schuell Inc., Keene, N.H.) usingthe Minifold II Slot Blotting System (Schleicher & Schuell Inc., Keene,N.H.). Standards of VEGF (2, 5, 10, 25 ng) (Sigma, Mississauga, ONT)were loaded on the same membrane. After drying at room temperature for10 minutes, the membrane was washed twice in TTBS buffer (50 mM Tris-HClbuffer pH 7.4 and 0.1% of Tween-20) for 5 minutes. Subsequently, themembrane was treated for 60 minutes with blocking buffer (BoehringerMannhein, GmbH, German). Monoclonal antibodies against VEGF (Sigma,Mississauga, ONT) (1:3000, diluted with 0.5×block buffer) were added andincubated overnight at 4° C. After washing twice with TTBS for 5 minuteseach, the membrane was incubated with goat anti-mouse-HRP conjugatedantibody (1:3000 dilution)(Bio-Rad Laboratories, Hercules, Calif.) for60 minutes at room temperature. The membrane was again washed twice withTTBS for 10 minutes twice. VEGF proteins were detected bychemiluminescence using the Boehringer Mannheim detection kit (Quebec,Canada). A densitometric analysis of the standard and sample bands wasperformed with a Bio-Rad image analysis system (Bio-Rad Lab. Hercules,Calif.). Standard curves of VEGF were generated and VEGF quantity inculture medium and cultured endothelial cells was calculated based onthe standard curve. The results (mean±SD) are expressed as ng of VEGF/mgprotein.

Preparation of Cells for Transplantation: Cultured endothelial cellswere passaged twice before transplantation to increase the number ofcells. Prior to transplantation, the cultured endothelial cells weredetached from culture dish with 0.05% trypsin in PBS. Aftercentrifugation in 580 g for 3 minutes, the cell pellets were resuspendedin culture medium at a concentration of 1.0×10⁶ cells/10 ul. A 60 ulcell suspension was used for each transplantation.

Myocardial Scar Formation: Rats were anesthesized by intramuscularinjection of ketamine hydrochloride (20 mg/kg body weight) followed byintraperitoneal injection of sodium pentobarbital (30 mg/kg bodyweight). The anesthesized rats were intubated and positive pressureventilation was performed with room air supplemented with oxygen andisoflurane (0.2-1.0%) using a Harvard ventilator. The electrocardiogramwas monitored during operation.

The heart was exposed through a 2- to 3-cm left lateral thoracotomyincision. Cryoinjury was produced in the left ventricular freewall(LVFW) with a metal probe (8×10 mm in diameter) cooled to −190° C.by immersion in liquid nitrogen. It was applied to the LVFW for oneminute and this procedure was repeated ten times. The muscle and skinwere closed with 3-0 silk sutures. Penlong XL (benzathine penicillin G150,000 U/ml and procaine penicillin G 150,000 U/ml) was givenintramuscularly (1 ml/kg) and buprenorphine hydrochloride (0.01 mg/kg)was administered after each operation. The cryoinjured rats wererandomly divided into two groups: transplantation and control.

Endothelial Cell Transplantation: At two weeks after cryoinjury, celltransplantation was performed. Under general anesthesia, the heart wasexposed through a midline sternotomy. Endothelial cell suspension (60ul, 6×10⁶ cells). was injected into the center of the scar tissue in thetransplantation group using a tuberculin syringe, and same amount ofculture media was injected into the scar in the control group. The chestwas closed with 3-0 silk sutures. Antibiotics and analgesics were givenas previously described. Cyclosporine A (25 mg/kg/day) was administeredsubcutaneously to both transplanted and control groups.

Identification of Transplanted Endothelial Cells: The transplanted cellswere identified in two ways.

(1) At day 1, 7 and 14 post transplantation, two rats from transplantedand control groups were sacrificed by exanguination under generalanesthesia. The hearts were fixed in with 10% phosphate-bufferedformalin solution for 2 days and cut into 3-mm sections. The sectionswere fixed in 5% glacial acetic acid in methanol, embedded in paraffin,and cut to yield 10 μm which were stained with hematoxylin and eosin asdescribed by the manufacturer (Sigma Diagnostics, St. Louis, Mo.).

(2) The transplanted cells also were identified by detection of greenfluorescent protein expressed by the transplanted cells. At 2 dayspre-transplantation, cultured endothelial cells werelipofectamine-transfected with a plasmid encoding green fluorescentprotein. The transfected cells were cultured, harvested and transplantedas described previously. At end of the study, monoclonal antibodiesagainst green fluorescent protein were used to localize the transplantedendothelial cells by avidin-biotin-peroxidase complex technique (Hsu, S.M. Am. J. Pathol., 75:816-821, 1981). Briefly, sections were incubatedwith a solution of 3% H₂O₂ in 70% methanol for 30 minutes to inhibitendogenous myocardial peroxidase. Nonspecific protein binding wasblocked with 2% normal goat serum in 0.05 M Tris buffer (pH 7.4) for 15minutes, primary antibodies against green fluorescent protein (CedarlaneLab., Hornby, Ontario) were added and the samples were incubated at 37°C. for 30 minutes followed by an overnight incubation at 4° C. Negativecontrol samples were incubated in PBS (without the primary antibodies)under the same conditions. After washing with PBS (three times for 5minutes each), a biotin-labeled secondary antibody (1:250, Vector Lab.Inc. Burilingame, Calif.) was added and the specimens were incubated atroom temperature for 1 hour. The samples were rinsed 3 times for 5minutes each in fresh PBS and reacted with an avidin-biotin complexconjugated with peroxidase at room temperature for 45 minutes.Visualization was performed with a diaminobenzidine solution (0.25 mg/mlin 0.05 Tris-HCl buffer containing 0.02% H₂O₂) for 10 minutes. Thesamples were then covered with crystal mounts and photographed.

Blood Flow Measurement with Radionuclide-labeled Microspheres: At sixweeks after transplantation, blood flow to the normal and scar tissue inthe transplanted (n=9) and control groups (n=8) was measured usingradionuclide-labeled microspheres (Pang, C.Y. Plastic and ReconstructiveSurgery 74(4):513-521, 1984). Rats were anesthesized and heparin sodium(200 units) was then administered intravenously. Hearts were excised andperfused in a Langendorf apparatus with 0.9% saline solution to wash outall blood cell remnants in the coronary vascular bed. Ten ml of 20 mEqKCl solution was injected through the coronary arteries to arrest theheart completely, and the heart was perfused with 0.9% saline solutionfor 10 minutes.

⁵⁷Co-labeled microspheres (5 uCi, New England Nuclear, Boston, Mass.)were suspended in 2 ml of 0.9% saline containing 5% sucrose and 0.05%Tween 80. The suspension was vortexed vigorously for 2 minutesimmediately before injection. The microspheres were infused over a 5second period through a needle connected to the ascending aortic root.

Ventricles were dissected into normal tissue, scar tissue, and tissue inthe borderline area. Each section was weighed and the radioactivity wasthen determined using a gamma counter at the window setting of 110 to138 KeV. The data was expressed as counts per minute (cpm)/mg of tissue.The ratio of cpm/mg in scar and borderline tissue to normal tissue wascalculated, expressed as the percentage, and compared betweentransplanted and control hearts.

Heart Function Measurement: Six weeks after transplantation, heartfunction in transplanted (n=10) and control (n=9) groups was measuredusing a Langendorf preparation (Stephen, S. E. Annals of ThoracicSurgery 59:1127-1133, 1995). Rats were anesthesized and heparin sodium(200 units) was administered intravenously. Hearts were quickly isolatedand perfused at 37° C. in a Langendorf apparatus with filteredKrebs-Hanseleit buffer (mmol/L: NaCl 118, KCl 4.7, KH₂PO₄ 1.2, CaCl₂2.5, MgSO₄ 1.2, NaHC0₃ 25, glucose 11, pH 7.4) equilibrated with 5%carbon dioxide and 95% oxygen at a pressure of 100 cm H₂O. A latexballoon was inserted into the left ventricle through the mitral valveand connected to a pressure transducer (Model p10EZ; Viggo-Spectramed,Oxnard Calif.) and a transducer amplifier and differentiator amplifier(Model 11-G4113-01; Gould Instrument System Inc, Valley View Ohio).

After 20 minutes of stabilization, the coronary flow was measured by thetimed collection in the empty beating state. The balloon size wasincreased in 0.02 ml increments from 0.04 to 0.6 ml by the addition ofsaline solution. The systolic and end-diastolic pressure and maximal andminimal dp/dt were recorded at each balloon volume. The developedpressure was calculated as the difference between the systolic andend-diastolic pressure.

Measurement of Left Ventricular Remodeling: The epicardial andendocardial surfaces of the normal and scar tissue in the LVFW weremeasured by the technique of Pfeffer (Pfeffer Am J Physiol1991;260:H1406-14) and Jugdutt and Khan Circulation 1994;89:2297-307).Briefly, the hearts were fixed in distension (30 mmHg) with 10%phosphate-buffered formalin solution for 2 days. The atria were excised,the ventricular weight and the size of the scar on epicardial surfacewere measured, and ventricular volume was measured by waterdisplacement. After that, the hearts were cut into sections of 3 mmthickness. For each section, the area of normal and scar tissue in theLVFW were traced onto a transparency and quantified using computedplanimetry (Jandal Scientific Sigma-Scan, Corte Madera, Calif.). Thelengths of LVFW and scar tissue on both the endocardial and epicardialsurfaces of each section were measured. The surface areas of theendocardial and epicardial scar tissue and the LVFW were measured as thesum of the endocardial length and epicardial length times the sectionthickness (3 mm). The surface area percentage of scar tissue in the LVFWwas calculated as follows: (endocardial scar size+epicardial scarsize)/(endocardial LVFW+epicardial LVFW)×100.

Measurement of Capillaries in Scar Tissue: The heart sections were usedfor immunohistochemical staining of endothelial cells. The sections wereincubated with xylene twice for 5 minutes each, 100% ethanol twice for 2minutes each, and then with 70% ethanol twice for 1 minute each. Thesections were incubated with rabbit immunoglobulin G against factorVIII-related antigen and endothelial cells were identified as describedin the Cell Identification section above. The stained vasculoendothelialcells in the transplanted and control groups were counted using a lightmicroscope at 400× magnification. The result was expressed as the numberof capillary vessels/field.

Syngeneic endothelial cell transplantation: To evaluate angiogenesisinduced by transplanted endothelial cells in the absence of aninflammatory reaction, endothelial cells isolated from adult Lewis rataorta were transfected and transplanted into myocardial scar tissue ofsyngeneic rat hearts (n=5) two weeks after the scarring procedure. Thetransplanted cells were identified in the scar tissue at 6 weeks aftertransplantation by immunohistochemical staining. The number ofcapillaries in the scar areas of transplanted and control hearts wascounted and compared. All the procedures performed on these animals weresame as described above, except that cyclosporine was not administeredafter cell transplantation.

Statistical Analysis: Data were expressed as the mean±standard error.Statistical Analysis System software was used for all analysis (SASInstitute, Cary, N.C.). Student's t-test was used for comparison of theresults. Cardiac function data were evaluated for the transplantationand control groups using intracavitary volume as the variant factor andsystolic, end-diastolic, and developed pressure as dependent variables.

Results

Cultured endothelial cells were distinguished from fibroblasts andvascular smooth muscle cells by morphologic criteria and growthcharacteristics. The endothelial cells were oval shaped, in contrast tothe spindle-shape of cultured fibroblasts and vascular smooth musclecells. Cultured endothelial cells grew in a “cobblestone” patternwhereas fibroblasts grew in a “whirling” pattern and vascular smoothmuscle cells grew in a “hill and valley” pattern. In addition, culturedendothelial cells stained positively for factor VIII-related antigen.The purity of the culture to be transplanted was more than 95%.

We found that cultured vascular endothelial cells contained VEGF (0.007and 0.005 ng/μg of cell protein, N=2). The VEGF molecules in thecultured cells were secreted into cultured medium: VEGF protein levels(0.331 and 0.229 ng/50 μl, N=2) in conditioned medium (i.e., in whichcells were cultured) was greater than in medium not exposed toendothelial cells (0.143 ng/50 μl, N=1).

At one day after transplantation, two rats in the transplanted groupwere sacrificed in order to identify transplanted cells. A largeendothelial cell cluster was observed within each scar. There was noincreased capillary network around the cell clusters compared withcontrol rats.

At one week after transplantation, there were some capillaries aroundthe transplanted endothelial cell cluster within each scar. The size ofthe endothelial cell cluster was, however, much smaller than thatobserved one day after transplantation.

At two weeks after transplantation, there were more capillaries withinthe scar but the endothelial cell cluster was no longer present.

Six weeks after transplantation, the number of blood vessels in the scartissue of the transplanted group (3.4±2.0 vessels/0.8 mm², n=10) wassignificantly greater (p<0.01) than within control (non-transplanted)scars (1.0±0.6, n=9). Most of blood vessels are capillaries withoutsmooth muscle components. In agreement with the number of blood vessels,the blood perfusion (represented as microsphere count) in the scartissue of transplanted hearts (6.85±2.27% of normal myocardium) wasgreater (p<0.01) than that of control hearts (3.78±0.99%) (FIG. 2).

In syngeneic cell transplantation the transplanted cells also inducedangiogenesis in the myocardial scar tissue. The density of capillariesin the scar tissue of the transplanted group was 2.6±1.5 vessels/0.8mm², which was significantly (p<0.01) greater than that in the controlgroup (1.1±0.6). Transplanted endothelial cells were incorporated intocapillaries at the transplant site.

In morphological studies of the hearts, there was no difference inweights of heart, left ventricle, scar tissue, and animal body betweentransplanted and control groups (Table 1). Ventricular volume intransplanted hearts was similar to that of control hearts. The size ofthe scar tissue in transplanted group was also not different from thatof control group.

There were no statistically significant differences in systolic anddeveloped pressures between transplanted and control hearts (FIGS. 3 and4), and no differences in coronary flow and heart rate (Table 1). TABLE1 Hemodynamics and heart dimensions in transplantation and controlgroups^((a,b)) Coronary flow Heart rate Ventricular weight Ventricularvolume Scar size Group (ml/min) (bpm) (g) (ml) (mm²) Transplant 20.9 +/−4.36 279 +/− 15 1160 +/− 168 1.86 +/− 0.27 109 +/− 19 Control 21.9 +/−3.82 289 +/− 19 1150 +/− 137 1.79 +/− 0.21 110 +/− 19^(a)Data are shown as the mean +/− standard error^(b)There were no significant differences in all parameters between twogroups.

The transplanted endothelial cells stimulate angiogenesis, demonstratedby an increase in the number of capillaries in the scar tissue andincreased blood perfusion in scar tissue as illustrated by microsphereperfusion studies. However, the induced angiogenesis in transmural scartissue did not improve infarcted heart function.

There are three possible mechanisms of increased angiogenesis byendothelial transplantation: (1) formation of blood vessels bytransplanted endothelial cells; (2) angiogenesis stimulated by growthfactors secreted by transplanted endothelial cells; (3) angiogenesisstimulated by an inflammatory reaction induced by transplantedendothelial cells.

Endothelial cells are the most important component for blood vesselformation. Our in vivo study showed that transplanted endothelial cellsbecome part of newly formed capillaries in syngeneic celltransplantation. These data suggested that transplanted endothelialcells are involved in blood vessel formation. However, the data obtainedfrom allogeneic endothelial cell transplantation suggested that newlyformed capillaries in the scar tissue were grown from recipient heartendothelial cells. We found transplanted cells at 1 and 7 days aftertransplantation, but did not observe transplanted cells after 14 dayspost transplantation. Additional evidence of this conclusion is thatthere was no immunologic reaction around capillary vessels in the scar.If the transplanted allogenic endothelial cells become part of the newlyformed capillaries, lymphocyte infiltration should be observed aroundthe blood vessel. These data illustrate that transplanted endothelialcells are involved in new blood vessel formation and also stimulateangiogenesis. In the latter situation, angiogeneic factors secreted bytransplanted cells may play an important role in this process.

Angiogenesis in our animal model might also be stimulated byinflammatory reactions. Although syngeneic endothelial celltransplantation has shown that angiogenesis occurrs without inflammatoryreaction, factors released by inflamatory cells might play a role inblood vessel formation induced by allogenic endothelial celltransplantation. Capillary density in scar tissue subjected to allogeniccell transplantation was 1.3 time greater than that subjected tosyngeneic cell transplantation. This difference in capillary density maybe due to lymphocyte infiltration.

Although blood flow was increased by endothelial cell transplantation inour experimental model, increased blood flow did not have any effects onleft ventricular remodeling and function. We found muscle cells in thescar tissue of a few hearts but the number of the muscle cells may notbe sufficient to improve heart function. There were no differences inheart volume, heart weight, and scar size between transplanted andcontrol hearts. In the analysis of function, there were no differencesin heart rate, coronary flow, systolic and developed pressures betweentwo groups.

We observed that the 2 week old scar was less mature and contained moreblood vessels and fibroblasts than 4 week old scar, which was firmconnective tissue. In addition, we found that cardiomyocytetransplantation was most successful after the inflammatory reactionresolved and before significant scar expansion and ventriculardilatation occurred.

The finding of increased angiogeneis in transmural scars induced byendothelial cell transplantation gives rise to a new technique forinduction of angiogenesis. We observed an increased number of capillaryvessels within the scars of rat hearts that were transplanted withendothelial cells. In contrast, we observed an increased number ofarterioles and venules in scars transplanted with fetal cardiomyocytes.If endothelial cells and cardiomyocytes are co-transplanted intomyocardial scars and endothelial cells augment the blood flow into thetransplanted area, the survival and function of transplantedcardiomyocytes might be enhanced. It would be possible to obtainventricular cardiomyocytes from transvenous endomyocardial biopsy andendothelial cells from saphenous vein or omental fat and culture thosecells for subsequent auto co-transplantation.

In addition to morphologic evidence of increased angiogenesis, wedemonstrated that blood flow to scar tissue was increased. Because therewas usually adhesion between the scar area and the chest wall from theprevious two operations (scarring and transplantation), blood flow tothe scar area may result from two sources, one from coronary arteriesand the other from the chest wall by non-coronary collateral vesselsproduced by adhesion. If blood flow from chest wall adhesion is countedtogether with blood flow from coronary artery, it obscures the effect ofangiogenesis per se on blood flow to the scar area. Therefore, weinjected microspheres into isolated heart preparations, although foractual blood flow estimation, injection of microspheres into left atriumor left ventricle in vivo may be more physiologic. Because there wereareas of non-transmural infarction in the periphery of the cryoinjuredlesions, the left ventricular free wall was divided into three parts:normal area, transmural infarct area, and borderline area. The bloodflow to the borderline areas was always between that of the normal andcomplete transmural infarct area.

Optimal Time for Cardiomyocyte Transplantation for Maximizing MyocardialFunction after Left Ventricular Necrosis

After myocardial infarction, damaged myocardium undergoes both acute andchronic inflammatory reactions. During this process the necrosed cardiaccells are replaced with fibrotic tissue and ventricular remodelingprogresses. Ventricular pressure stretches and thins the healing areaand ventricular dilatation occurs. A ventricular aneurysm may form andcongestive heart failure may result. Limitation of post infarctionventricular remodelling is important to prevent expansion and thinningof the infarct region and thinning, and progressive ventriculardilatation, which results in congestive heart failure. The addition ofcardiac tissure to replace the scar tissue may prevent infarct thinningand preserve chamber size and ventricular function. We had shown thatfetal cardiomyocytes transplanted into a LVFW scar formed cardiac tissueand improved myocardial function. Transplantation was arbitrarilyperformed at 4 weeks after cryo-necrosis of the LVFW when ventriculardilatation was already present. However, the optimal time forcardiomyocyte transplantation to provide the best ventricular functionis important. The acute inflammatory reaction could destroy thetransplanted cardiomyocytes that are introduced too soon aftermyocardial infarction. On the other hand, cardiomyocyte transplantationafter significant scar expansion may be of limited benefit. Therefore,the present study investigated the optimal time for cell transplantationafter myocardial injury.

Summary

Methods:

(1) Scar expansion studies: The rats were sacrificed at 0, 1, 2, 4, and8 weeks after cryo-injury and the scar area was measured by planimetry(N=7 each).

(2) Transplantation studies: Fetal rat cardiomyocytes (transplant) orculture medium (control) were transplanted immediately (N=8 each), 2weeks (N=8 each) and 4 weeks (N=12 each) after cryo-injury. At 8 weeksafter cryo-injury, rat heart function was evaluated using a Langendorffpreparation. Scar and transplanted cardiomyocytes were assessedhistologically.

Results:

(1) A transmural injury occurred immediately after cryo-injury. Aninflammatory reaction was more evident at 1 week than 2 weeks. Althoughthe scar size did not significantly change between week 1 and 2, itexpanded (p<0.01) at 4 and 8 weeks.

(2) At 8 weeks after myocardial injury, cardiomyocytes transplantedimmediately after myocardial injury were not found in the myocardialscar tissue. Scar size and myocardial function were similar to those ofthe control hearts. Cardiomyocytes transplanted at 2 and 4 weeks formedcardiac tissue, which occupied 34% and 31% of total scar area,respectively, and limited (p<0.01) scar expansion compared to controlhearts. Both transplant groups had better (p<0.001, <0.001) heartfunction than the control groups. Developed pressure was greater(p<0.01) in the hearts transplanted cells at 2 weeks than at 4 weeks.

Conclusion:

Cardiomyocyte transplantation was most successful after the inflammatoryreaction resolved and before significant scar expansion and ventriculardilatation occurred.

Material and Methods

Experimental animals: All procedures performed on animals were approvedby the Animal Care Committee of The Toronto Hospital. Experimentalanimals used were male Sprague-Dawley rats (Lewis, Charles River CanadaInc. Quebec, Canada), weighing 450 grams. Cardiomyocytes obtained from18-day gestational rat hearts were cultured prior to transplantation.All experiments were performed according to “A Guide to the Care and Useof Experimental Animals” of the Canadian Council on Animal Care and the“Guide for the Care and Use of Laboratory Animals” NIH publication85-23, revised 1985).

Myocardial scar generation and evaluation: Myocardial scar tissue wasgenerated as described in Example I above. Briefly, rats wereanesthetized with ketamine (22 mg/kg body weight, intramuscular)followed by an intraperitoneal injection of pentobarbital (30 mg/kg bodyweight). The anesthetized rats were intubated and positive pressureventilation was maintained with room air supplemented with oxygen (6L/minute) using a Harvard ventilator (Model 683, South Natick, Mass.,USA). The rat heart was exposed through a two cm left lateralthoracotomy. Cryo-injury was produced at the left ventricular free wall(LVFW) with a liquid nitrogen probe. The muscle layer and skin wereclosed with 5-0 vicryl sutures. The rats were monitored for 4 hourspostoperatively. Penlog XL (benzathine penicillin G 150,000 U/ml andprocaine penicillin G 150,000 U/ml) were given intramuscularly (0.25ml/rat) every three days for 1 week after surgery and buprenorphine(0.01-0.05 mg/kg body weight) was administrated subcutaneously every 8to 12 hours for the first 48 hours following surgery.

The LVFWs of 35 animals were cryo-injured. Immediately after cryo-injuryand at 1, 2, 4, and 8 weeks after injury, 7 animals chosen at randomwere sacrificed under general anesthesia (see FIG. 5 for a schematicdiagram of the experimental design). The hearts were then fixed indistension (30 mm Hg) with 10% phosphate-buffered formalin solution for24-48 hours and then cut into 3 mm thick sections. All sections wereused for assessment of myocardial and scar sizes. For each section, thearea of scar tissue in the LVFW was traced onto a transparency andquantified using computerized planimetry (Jandal Scientific Sigma Scan,Fairfield, Conn., USA). Scar length was calculated as the average ofendocardial scar length and epicardial scar length. Scar area wascalculated as the scar length×section thickness (3 mm). The total scararea in size was calculated as the sum of the scar areas for all of thetissue sections showing scars. The LVFW myocardial size was calculatedusing the same equation. The percentage of the LVFW occupied by the scarwas calculated as scar size/LVFW size×100.

Histological studies: The fixed heart sections were embedded in paraffinand cut to yield 10 μm thick sections. The sections were stained withhaematoxylin and eosin as described by the manufacturer (SigmaDiagnostics, St. Louis, Mo.).

Cell culture and preparation for transplantation: Cardiomyocytes fromfetal rat hearts were isolated, purified and cultured as previouslydescribed (Li, R-K, et al. J. Tissue Culture Methods 14:93-100, 1992).Briefly, the cells were cultured for 24 hours in Iscove's modifiedDulbecco's medium containing 10% fetal bovine serum, 100 U/ml penicillinand 100 ug/ml streptomycin at 37EC, 5% CO₂ and 95% air. The culturedcardiomyocytes were detached from the cell culture dish with 0.05%trypsin in phosphate-buffered saline (PBS). After centrifugation at580×g for 3 minutes, the cell pellet was resuspended in culture mediumat a concentration of 16×10⁶ cells/ml. A 0.25 ml cell suspension wasused for each transplantation.

Cardiomyocyte transplantation: The LVFW of 56 adult rat hearts werecryo-injured as described above. The animals were randomly divided into3 groups (see FIG. 5, bottom). In group one, 8 animals were transplantedwith cultured cardiomyocytes (transplant) and 8 animals weretransplanted with culture medium (control) immediately after myocardialinjury. In group two, animals were transplanted with cells and culturemedium (N=8 each) 2 weeks after myocardial injury. In group three,control and transplanted animals (N=12 each) were transplanted 4 weeksafter myocardial injury. All animals were maintained 8 weeks after thetransplant procedure.

Under general anesthesia, the hearts were exposed through a midlinesternotomy. A fetal rat cardiomyocyte suspension (0.25 ml, 4×10⁶ cells)or culture medium (0.25 ml) was injected once into the center of thescar tissue of the transplanted and control animals, respectively, usinga tuberculin syringe. The chest was closed with 5-0 vicryl sutures.Antibiotics and analgesics were given as described in the “MyocardialScar Generation and Evaluation” section below. Cyclosporin A, at a doseof 5 mg/kg body weight/day, was administered subcutaneously to thecontrol and transplanted rats. The rats were housed in cages with filtertops.

Myocardial function studies: At 8 weeks after myocardial injury, theheart function of control and transplanted animals transplanted at 0,2,and 4 weeks after myocardial injury was measured using a Langendorffpreparation (Stephen, S. E. Annals of Thoracic Surgery 59:1127-1133,1995). The rats were anesthetized and heparin (200 units) wasadministered intravenously. The hearts were quickly isolated from ratsand perfused in a Langendorff apparatus with filtered Krebs Heinseleitbuffer (mmol/L NaCl 118, KCl 4.7, KH₂PO₄ 1.2, CaCl₂ 2.5, MgSO₄ 1.2,NaHCO₃ 25, glucose 11; pH 7.4) equilibrated with a 5% CO₂ and 95% O₂. Alatex balloon was passed into the left ventricle through the mitralvalve and connected to a pressure transducer (Model p10EZ,Viggo-Spectramed, Calif.) and transducer amplifier and differentiatoramplifier (Model 11-G4113-01, Gould Instrument System Inc., Ohio). After30 minutes of stabilization, the balloon size was increased in 0.02 mlincrements from 0.04 to a volume, at which end diastolic pressure was 30mm Hg or more by the addition of saline. The systolic and diastolicpressures were recorded at each balloon volume and developed pressurewas calculated as the difference between the systolic and diastolicpressures.

Measurement of left ventricular free wall remodeling: After functionmeasurements, the hearts were fixed, sectioned, and traced onto atransparency. LVFW and scar sizes were quantified using computerizedplanimetry as described in the “Myocardial Scar Generation andEvaluation” section. The transplanted tissue size as a percentage of thescar size was similarly calculated.

Histological identification of transplanted muscle cells in the scartissue: Fixed heart sections were embedded in paraffin, and cut into 10μm thick sections. The sections were stained with hematoxylin and eosinas described in the “Histological Studies” section above.

Heart sections containing transplanted cells also were stained formyosin heavy chain (MHC) (Rougier Bio-Tech, Montreal). Afterdeparaffination and rehydration, samples were incubated for 30 minuteswith a solution of 3% H₂O₂ in 70% methanol to inhibit endogenousmyocardial peroxidase. Triton X-100 (0.2%) was used to treat samples for10 minutes to enhance cell permeability. After blocking nonspecificprotein binding with 2% normal goat serum in 0.05 M Tris buffer (pH 7.4)for 15 minutes, primary antibodies against HCM (1:1000) were added andthe samples were incubated at 37° C. for 30 minutes followed by anovernight incubation at 4° C. Negative control samples were incubated inPBS under the same conditions. After samples were washed with PBS, abiotin-labeled secondary antibody (1:250) was added and the samples wereincubated at room temperature for 1 hour. After rinsing with PBS, thesamples were exposed to an avidin-biotin complex conjugated withperoxidase at room temperature for 45 minutes. Visualization wasperformed with a diaminobenzidine solution (0.25 mg/ml in 0.05 Tris-HClbuffer containing 0.02% H₂O₂) for 10 minutes. The cellular nuclei werecounter-stained with hematoxylin for 1 minute. The samples were coveredand photographed.

Data Analysis: Data are expressed as the mean±standard error. TheStatistical Analysis System software was used for all analysis (SASInstitute, Cary, N.C.). Comparisons of continuous variables between morethan two groups were performed by a one-way analysis of variance. If theF ratio was significant from the ANOVA, a Duncan=s multiple-range t testwas employed to specify differences between the groups. The criticalalpha-level for these analyses was set at p<0.05.

Functional data were evaluated for the control and transplant groups byan analysis of covariance (ANCOVA) using intracavitary balloon volume asthe covariate and systolic, diastolic and developed pressure asdependent variables. Main effects were group, volume, and theinteraction between group and volume.

Results

Histological and morphological changes of the hearts after myocardialinjury: Immediately after cryo-injury, 25±3% of the size of the LVFW wastransmurally damaged. The cardiomyocytes were fragmented (FIG. 6A). Atone week, most of the necrosed cardiomyocytes were gone and apredominantly mononuclear inflammatory infiltrate was present in theaffected area (FIG. 6B). At two weeks the inflammatory infiltrate hadalmost disappeared and fibroblasts and collagen deposition were evident(FIG. 6C). At four and eight weeks, the scar was composed of fibrotictissue (FIG. 6D). The tissue was less cellular and lymphocytes were notobserved.

The myocardial scar size of the left ventricle expanded over the 8 weekstudy period in the damaged hearts (FIGS. 7 and 8). Although the scarsizes at 1 and 2 weeks (13±6% and 21±4 of LVFW) were not statisticallydifferent, the size of 4-week-old scars (39±5% of LVFW) was larger(p<0.01). At 8 weeks there was a further increase (p<0.0) in scar size(55±3% of LVFW).

Although histological studies showed that the damage tonewly-injuredmyocardium was transmural, some myocardium (12±5% of totalscar area) was observed at the endocardium one week after myocardialinjury (FIGS. 6 and 7). Lymphocytes surrounded the myocardium at thistime. At 2 and 4 weeks, muscle tissue in the scar area (3±2% and 2±1.5%of total scar area) decreased dramatically (p<0.01) in size. At 8 weeks,the myocardial scar became transmural and there was no myocardium in thescar tissue.

Effect of transplanted cells on scar size: The fetal rat cardiomyocytestransplanted into myocardium immediately after myocardial damage did notsurvive in the transplanted area (FIGS. 9 and 10). The scar area (53±5%)of transplanted hearts was similar to that of the control group 55±3% ofthe LVFW. There was no cardiac tissue at endocardial area.Cardiomyocytes transplanted at 2 weeks after myocardial damage formedcardiac tissue which stained positively for myosin heavy chain (FIG.11). The newly formed cardiac tissue occupied 34% of the total scar area(11±3% of the LVFW). Similarly cardiomyocytes transplanted at 4 weeksoccupied 31% of total scar area (12±2% of LVFW).

In addition to the cardiac tissue formed by transplanted cardiomyocytes,recipient myocardium was also found at endocardium in both transplantedgroups. The scar sizes for both the 2 and 4 week hearts transplantedwith cells were smaller (p<0.0) than the scar size of the control(nontransplantable) hearts. The scar size of the hearts transplanted at2 weeks (32±5%) was smaller (p<0.01) than that of the heartstransplanted at 4 weeks (42±2%) (FIG. 10).

Effect of transplanted cells on heart function: When cardiomyocytes weretransplanted immediately after myocardial injury, ventricular functionof hearts transplanted with cells was similar to that of control(nontransplanted) hearts (FIG. 12). An analysis of covariancedemonstrated no association between the control group and the treatmentgroup for developed pressures. When the cardiomyocytes were transplantedat 2 weeks after myocardial injury, the transplanted hearts had better(p=0.001) ventricular function than the control hearts (FIG. 13).Similar diastolic and developed pressures were found at lower balloonvolumes in the transplant group.

Cardiomyocytes transplanted at 4 weeks after myocardial injury alsoimproved (p<0.001) myocardial function (FIG. 14). FIG. 15 shows thathearts transplanted at 2 weeks had higher developed pressures at balloonvolumes 0.1 ml (p<0.05), 0.2 ml (p<0.01) and 0.3 ml (p<0.01) than heartstransplanted at 4 weeks.

The transplant tissue visibly contracted, but at different rates thandid host myocardium. The contraction of the transplanted tissuepersisted after dissection of the recipient heart, but its rate ofcontraction continued to be different.

In the rat, loss of necrosed ventricular tissue occurs by 2 days aftercyro-injury of the myocardium. At this time there is an acuteinflammatory reaction with neutrophils accumulating first in theperiphery and later in the center of the necrotic region. A chronicinflammatory reaction involving macrophages and lymphocytes follows theacute reaction.

One week after cryo-injury, more than 80% necrotic muscle fibres haddisappeared. The remaining muscle fibres were surrounded by lymphocytes.

The inflammatory reaction is less intense two weeks after cryo-injury(and is absent by three weeks): two weeks after cryo-injury, the scarsof the control animal hearts showed minimal or no inflammatory reaction.Two-week-old scars were less mature and contained more blood vessels andfibroblasts than 4-week-old scars, which comprised firm connectivetissue. Compared to one-week-old scars, there was less cardiac tissue inolder scars. It is possible that progressive inflammation and lack ofoxygen and nutrient supply decreased the myocardial tissue over time.

The cardiomyocytes transplanted immediately after cryo-injury did notsurvive in any of the animals studied. We believe the activatedneutrophils and macrophages in the inflamed scar area destroyed thetransplanted cells. At 8 weeks, the left ventricular chamber of theanimals transplanted at the time of cryo-necrosis was similar to that ofthe control scar. The chamber was dilated due to thinning of the scar.The remaining viable muscle of the left ventricle was hypertrophied. Noinflammatory reaction was seen. The function of these transplantedhearts was similar to that of the control hearts.

All the animals transplanted with the fetal cardiomyocytes at 2 and 4weeks formed cardiac tissue within the scar tissue; therefore,transplantation is possible in both the immature and mature scar.Histological studies of the LVFW of the transplanted hearts showed thathost myocardium was usually present beneath the scar when the transplantwas present. This did not occur when the transplant was absent.

Control scar studies showed that scars were always transmural and didnot have host cardiomyocytes underneath. Scar sizes and left ventricularchamber sizes of the hearts transplanted at 2 weeks were smaller thanthose of hearts transplanted at 4 weeks. In turn, scar sizes and leftventricular chamber sizes of the hearts transplanted at 4 weeks weresmaller than those of the control hearts. Consistent with thesehistological findings, the myocardial function of the heartstransplanted at 2 weeks was better than that of the hearts transplantedat 4 weeks. We attributed the improved myocardial function to lessventricular dilatation and scar thinning. Compared with thesham-operated animals in our previous study, the contractile function ofthe hearts transplanted at 2 weeks was less efficient. Transplantationas soon as possible after the acute inflammatory reaction in theinfarcted myocardium has disappeared should minimize ventricularremodeling and optimize myocardial function.

Although the mechanism of improved heart function is unknown, acardiomyocyte transplant forms the equivalent of a viable epicardial rimand prevents ventricular dilatation and over-stretching of thecardiomyocytes during systole. With over-stretching of thecardiomyocytes, cardiac function is diminished (Frank-Starling Law). Theelastic properties of the contractile apparatus of the transplantedcardiomyocytes may prevent host fibroblast and cardiomyocyte stretchingand ventricular enlargement.

In the course of forming cardiac tissue from transplantedcardiomyocytes, angiogenesis occurred. Angiogenesis is most likelynecessary to maintain the viability of the transplanted muscle cells.However, an increased blood supply in the scar could also facilitatefibroblast turnover and strengthening of the scar in response to leftventricular free wall stretch.

With the advent of thrombolysis and percutaneous transluminal coronaryangioplasty (PTCA), the incidence of patchy nontransmural infarction isbecoming more frequent and transmural infarction with aneurysm formationless common. If the blood flow to the viable cardiomyocytes within theventricular scar is insufficient for contractile function, stimulationof angiogenesis in the scar tissue becomes important. With adequateblood flow, normal contractile function should return to the hibernatingcardiomyocytes within the scar. Transplantation of fetal cardiomyocytescould offer significant benefits to the patients with a patchyinfarction. Angiogenesis would be stimulated in the scar, increase theblood flow, and restore normal contractile function to the host'shibernating cardiomyocytes. The transplanted cardiomyocytes should alsoincrease the contractile capacity of the scar tissue and decrease thecontractile requirements of the non-affected host myocardium. Patientswith angina that cannot be corrected by bypass surgery may benefit fromangiogenesis secondary to cardiomyocyte transplantation. In addition, itmay be beneficial to stimulate angiogenesis with growth factors prior toautotransplantation.

In summary, transplantation of fetal cardiomyocytes form cardiac tissuein scar tissue which limits ventricular dilatation and scar thinning.The optimal time for transplantation for improved myocardial function isafter the acute inflammatory reaction and before significant ventriculardilatation has occurred.

Co-Implantation of Endothelial Cells with Adult Cardiomyocytes Improvesthe Function of Infarcted Hearts

Cardiac transmural scars were induced by cryo-injury in the leftventricular free wall (LVFW) of Sprague-Dawley rat hearts, as describedin the previous examples. Three weeks after cryo-injury, the rats weredivided into three groups (eight rats per group), and cardiac transmuralscars were transplanted with either medium (no cells), adultcardiomyocytes alone, or adult cardiomyocytes plus endothelial cells.Cardiac function was assessed using a Langandorff preparation five weeksafter cell transplantation (Stephen et al., Annals of Thoracic Surgery59: 1127-1133, 1995).

FIG. 16 shows a graph of developed pressure in infarcted rat heartsinjected with either medium (negative control), adult cardiomyocytesalone (CM), or adult cardiomyocytes plus endothelial cells (CM+EC).Adult cardiomyocytes injected alone formed tissue within the ventricularscar, but did not improve cardiac function, compared to hearts injectedwith medium alone. In contrast, injection of adult cardiomyocytes plusendothelial cells significantly improved cardiac function (p<0.01),compared to hearts injected with medium or cardiomyocytes alone.Co-injection of endothelial cells likely stimulates angiogenesis, whichenhances survival of adult cardiomyocytes.

Although the invention has been described with reference to certainspecific embodiments, various modifications thereof will be apparent tothose skilled in the art without departing from the spirit and scope ofthe invention as outlined in the claims appended hereto.

1-6. (canceled)
 7. A method of forming a stable cardiac graft in amammal, said method comprising transplanting adult fibroblasts into scartissue in said mammal's heart, wherein said transplanted fibroblastsimprove heart function in said mammal.
 8. The method of claim 1, whereinsaid graft is used for cardiomyoplasty.
 9. The method of claim 1,wherein said graft is used for closing cardiac defects.
 10. The methodof claim 1, wherein said graft is used for myocardial reconstructivesurgery.
 11. A method for improving heart function in a mammal withmyocardial injury, comprising the step of transplanting adultfibroblasts into said mammal's heart, wherein said fibroblasts aretransplanted into said mammal's heart after fibrotic tissue has formedin response to said injury, and wherein said transplanted fibroblastsimprove heart function in said mammal.
 12. A method for improving heartfunction in a mammal with heart damage due to acute myocardial injury,comprising the step of transplanting adult fibroblasts into saidmammal's heart, wherein said fibroblasts are transplanted into saidmammal's heart after an inflammatory response to said acute myocardialinjury has subsided and wherein said transplanted fibroblasts improveheart function in said mammal.
 13. The method of any one of claims 7,11, or 12, wherein said fibroblasts are autologous to the mammal beingtreated.
 14. The method of any one of claims 7, 11, or 12, wherein saidfibroblasts are allogeneic to the mammal being treated.
 15. The methodof any one of claims 7, 11, or 12, wherein said fibroblasts areadministered by direct injection into said mammal's heart.
 16. Themethod of claim any one of claims 7, 11, or 12, wherein said fibroblastsare transfected to deliver recombinant molecules to said mammal's heart.17. The method of claim any one of claims 7, 11, or 12, wherein saidfibroblasts are supported on a biodegradable or non-biodegradablescaffolding.
 18. The method of claim any one of claims 7, 11, or 12,wherein said fibroblasts are obtained from skin biopsy.
 19. The methodof claim 18, further comprising culturing said fibroblasts prior totransplanting.